Mitsuko Maeda

PEGylated adenovirus vectors containing RGD peptides on the tip of PEG show high transduction efficiency and antibody evasion ability

PEGylation of adenovirus vectors (Ads) is an attractive strategy in gene therapy. Although many types of PEGylated Ad (PEG‐Ads), which exhibit antibody evasion activity and long plasma half‐life, have been developed, their entry into cells has been prevented by steric hindrance by polyethylene glycol (PEG) chains. Likewise, sufficient gene expression for medical treatment could not be achieved.

Amino acids and peptides. Part 39: A bivalent poly(ethylene glycol) hybrid containing an active site (RGD) and its synergistic site (PHSRN) of fibronectin

Fibronectin contains the active sequence Arg-Gly-Asp (RGD), along with its synergic site Pro-His-Ser-Arg-Asn (PHSRN). However, the PHSRN peptide does not show synergic activity when it is mixed with the RGD peptide, indicating that a spatial array between RGD and PHSRN in fibronectin may be necessary for synergic activity. Here, we have used an amino acid type poly(ethylene glycol) derivative (aaPEG) to design a bivalent PEG hybrid of fibronectin active peptides. We prepared the aaPEG hybrid peptides PHSRN-aaPEG, aaPEG-RGD, and PHSRN-aaPEG-RGD, and tested their biological activity. Whereas aaPEG-RGD promoted cell spreading activity, PHSRN-aaPEG had no activity. The PHSRN-aaPEG-RGD hybrid strongly promoted cell spreading compared with aaPEG-RGD. These results suggest that the PHSRN sequence in the PHSRN-aaPEG-RGD molecule synergistically enhances the cell spreading activity of the RGD sequence, and that the bivalent aaPEG hybrid method may be useful for conjugating functionally active peptides.

Transduction of adenovirus vectors modified with cell-penetrating peptides

Adenovirus vectors (Advs) are widely used for basic and clinical research because of their high transduction efficiency. However, they are poorly transduced into cells lacking the primary adenovirus receptor, the coxsackievirus and adenovirus receptor (CAR). Here, we generated Adv conjugated with cell-penetrating peptides (CPPs), such as Tat, octaarginine (R8) or proline-rich (Pro) peptides, and compared the transduction properties of these constructs. We constructed the Advs conjugated to the CPPs (CPP-Adv) by chemical conjugation. The CPP-conjugated Advs created with optimal modification ratios led to gene expression 1-2log orders higher than unmodified Adv in CAR-negative cells. Tat-Adv and R8-Adv were taken up into the cells mainly through macropinocytosis, independently of the CAR. In addition, the cellular uptake of Tat-Adv was highly dependent on heparan sulfate on the cell surface, whereas that of R8-Adv was dependent on chondroitin sulfate B. These data suggest that the use of CPP-Advs with different cellular uptake pathways might create new methods for the delivery of Adv. The results obtained in this research encourage the use of CPP-peptide-modified Advs as an attractive tool for transducing cells and as useful platform vectors for gene therapy and basic research.

Facile synthesis of a chitosan hybrid of a laminin-related peptide and its antimetastatic effect in mice

Laminin, a cell adhesion protein, consists of three peptide chains (α, β and γ‐1). The β chain contains a Tyr‐Ile‐Gly‐Ser‐Arg (YIGSR) sequence that has been found to inhibit experimental metastasis in mice. We have prepared a hybrid of a water‐soluble chitosan and a laminin‐related peptide, and have examined its inhibitory effect on experimental metastasis in mice.

Antimetastatic effect of synthetic Glu-Ile-Leu-Asp-Val peptide derivatives containing D-amino acids.

The aim of this study was to increase the antimetastatic potency of the fibronectin-related peptide, Glu-lle-Leu-Asp- Val (EILDV), and to determine the minimal core sequence of EILDV required to inhibit tumor metastasis in vivo. The EILDV subpeptide analog, ILDV, markedly inhibited the adhesion of B16-BL6 melanoma cells to fibronectin. EILD and ILD were only slightly inhibitory, and the smaller overlapping tripeptide, LDV, was inactive. The inhibitory activities of ILDV and LDV on the migration of B16-BL6 melanoma cells were as potent as those of EILDV, whereas ILD did not inhibit cell migration. These results suggested that the minimal sequences essential for cell adhesion and migration are ILD and LDV, respectively. However, the antimetastatic effects of all subpeptide analogs were lower than that of EILDV. In order to improve the stability in vivo, we synthesized various EILDV-related peptides substituted with a D-amino acid. EILDV containing D-Glu or D-lle inhibited cell adhesion and migration as potent as EILDV, whereas replacing Leu, Asp or Val with the corresponding D-isomer reduced the antiadhesive activities. The inhibitory effect of EILDV-related peptides containing D-Leu, D-Asp or D-Val on migration was also lower than that of EILDV. All synthetic EILDV-related peptides containing D-amino acids inhibited metastasis by B16-BL6 melanoma cells to the same extent as EILDV, whereas the specific activity of EILDV was decreased by the D-amino acid substitution. These results indicated that the balance of stability in vivo and biological activity in vitro is important in inhibiting tumor metastasis

Tat conjugation of adenovirus vector broadens tropism and enhances transduction efficiency.

AIMS Adenovirus vectors (Advs) have been very useful for basic research and clinical gene therapy because they propagate to high titers and efficiently transduce cells and tissues regardless of the mitotic status. However, poor transduction of cells that lack the coxsackievirus and adenovirus receptor (CAR), the primary receptor for Advs, has limited Adv application. In this study, we attempted to generate novel Tat-Advs (Advs conjugated with the HIV Tat-derived peptide, a protein-transduction domain (PTD)) to broaden Adv tropism and enhance transduction efficiency. MAIN METHODS We constructed Tat-Advs by chemically conjugating Tat peptide to the surface-exposed lysine residues on Advs. We compared the gene transfer activity of Tat-Advs with that of unmodified Advs by measuring the luciferase expression in several types of cell lines. KEY FINDINGS Tat-Advs showed gene expression 1 to 3 log orders higher than unmodified Advs in CAR-negative adherent cells and blood cells, which are refractory to conventional Advs. The inhibition of Tat-Adv-mediated gene expression by heparin and macropinocytosis inhibitor confirms that binding of Tat-Adv to cellular HSPGs and macropinocytosis are essential for efficient CAR-independent transduction. We also demonstrated that Adv modified with another PTD (R8) had the same high transduction efficiency as Tat-Adv. SIGNIFICANCE These data suggest that Tat-Advs are important tools for transducing cells and will be useful as platform vectors for gene therapy.

Antimetastatic effects of synthetic peptides containing the core sequence of the type III connecting segment domain (IIICS) of fibronectin.

The antimetastatic activities of synthetic peptides corresponding to fragments of the adhesion-related molecules, such as flbronectin and laminin, were examined. We prepared three peptides derived from the type III connecting segment domain (IIICS) of flbronectin: Glullo-Leu-Asp-Val (EILDV), Qlu-lle-Leu-Aap-Val-Pro-Ser-Thr (EILDVPST), Arg-Qlu-Aap-Val (REDV), and a lamlnln-re-lated peptide, Tyr-lle-Gly-Ser-Arg (YIGSR). Each peptide inhibited the experimental tumor metastasis of B16-BL6 melanoma, while EILDV had the strongest effect. The peptides conjugated with poly(ethylene glycol) (PEG) were more effective than the unmodified peptides in molar ratio terms. A mixture composed of PEG hybrids with EILDV, REDV and YIGSR significantly Inhibited tumor metastasis.

Thiol-containing peptide-hemin complexes as models of cytochrome P-450

Nine different synthesized thiol tetra- and penta-peptides containing Cys-Ser, Cys-Thr, Cys-His, Cys-Tyr and Cys-Cys in the N- and C-terminals are proposed as new models of apo-P-450. The peptide-hemin complexes in the oxidized (Fe(III] form in solution were characterized by their optical and EPR spectra and found to show hydroxylation activity like that of P-450 enzymes on acetanilide. Although the EPR properties of the complex containing Cys-His and all complexes in the presence of pyridine were similar to those of P-450, the optical properties of these complexes were not completely similar to those of P-450. Based on these results, the sixth heme coordination site of P-450 was discussed.

Preparation of a Chitosan Hybrid of an Antimetastatic Laminin‐related Peptide

A hybrid of chitosan and an antimetastatic laminin-related peptide was prepared. Acetyl-Tyr-Ile-Gly-Ser-Agr-βAla-OH (Ac-YIGSRβA-OH) was prepared by a solid-phase method and reacted with a water-soluble chitosan. Chitosan amino groups did not react with the peptide using diphenylphosphoryl azide, diisopropylcarbodiimide/1-hydroxybenzotriazole, water-soluble carbodiimide/1-hydroxy-benzotriazole, phosphazo, or 2-(1 H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) methods. A small spacer was therefore put between the peptide and the chitosan. tert-Butyloxycarbonyl-Gly (spacer) was reacted with chitosan by the TBTU method. After removal of the protecting group, the Gly-chitosan was coupled with Ac-YIGSRβA-OH by the water-soluble carbidiimide method to give Ac-YIGSRβAG-chitosan. The inhibitory effect of the peptide-chitosan hybrid on experimental metastasis in mice was not reduced, but actually potentiated, suggesting that chitosan may be used as a drug carrier for peptides.

Evaluation of synthetic cell-penetrating peptides, Pro-rich peptide and octaargine derivatives, as adenovirus vector carrier.

Two cell-penetrating peptides, a Pro-rich peptide derivative, acetyl-(Val-Arg-Leu-Pro-Pro-Pro)(3)-Gly-Cys amide, and an octaarginine derivative, acetyl-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Gly-Cys amide, were prepared by the solid phase method. Each peptide was coupled to the heterobifunctional cross-linking reagent, 6-maleimidohexanoic acid N-hydroxysuccinimide ester, and then conjugated to the Adenovirus vector containing luciferase gene. Peptide-modified Ad, as compared with wild-type Ad, exhibited excellent luciferase activity in B16BL6 cells.