Enhancement of gene delivery to cancer cells by a retargeted adenovirus

The inefficiency of in vivo gene transfer using currently available vectors reflects a major hurdle in cancer gene therapy. Both viral and non-viral approaches that improve gene transfer efficiency have been described, but suffer from a number of limitations. Herein, a fiber-modified adenovirus, carrying the small peptide ligand on the capsid, was tested for the delivery of a transgene to cancer cells. The fiber-modified adenovirus was able to mediate the entry and expression of a beta-galactosidase into cancer cells with increased efficiency compared to the unmodified adenovirus. Particularly, the gene transfer efficiency was improved up to 5 times in OVCAR3 cells, an ovarian cancer cell line. Such transduction systems hold promise for delivering genes to transferrin receptor overexpressing cancer cells, and could be used for future cancer gene therapy.     

利用TAT多肽增强腺病毒对细胞感染效率的研究

蛋白转导多肽本身或携带生物大分子能以一种不明机制的方式高效地穿过真核细胞质膜并且几乎没有组织选择性。这为生物药物研究、基因治疗等领域带来了新的希望。最近有研究表明:来源于HIV-1的TAT蛋白的蛋白转导结构域多肽可以显著地提高重组腺病毒感染细胞和实验动物的效率。在对。HeLa且和Vero-E62种具有不同病毒易感性的细胞进行重组腺病毒感染实验时发现TAT多肽可以明显地提高重组腺病毒对HeLa细胞的感染及在细胞中外源报道基因的表达,但是对Vero-E6细胞却没有效果,表明TAT多肽增强重组腺病毒的感染与靶细胞类型有关,而并不像转导现象那样没有组织差异。这为蛋白转导技术在病毒载体中的应用提供了参考,但其中涉及的蛋白转导的机制有待进一步实验研究。     

Design and synthesis of a peptide-PEG transporter tool for carrying adenovirus vector into cells

The adenovirus vector is a promising carrier for the efficient transfer of genes into cells via the coxackie-adenovirus receptor (CAR) and integrins (alphavbeta3 and alphavbeta5). The clinical use of the adenovirus vector remains problematic however. Successful administration of this vector is associated with side effects because antibodies to this vector are commonly found throughout the human body. To make the adenovirus vector practicable for clinical use, it is necessary to design an auxiliary transporter. The present study describes the use of Arg-Gly-Asp(RGD)-related peptide, a peptide that binds to integrins, as an auxiliary transporter to aid efficient transport of adenovirus vector. Furthermore, poly(ethylene glycol) (PEG) was also used as a tool to modify the adenovirus such that the risk of side effects incurred during clinical application was reduced. The present study describes the design, preparation and use of (acetyl-Tyr-Gly-Gly-Arg-Gly-Asp-Thr-Pro-(beta)Ala)(2)Lys-PEG-(beta)Ala-Cys-NH(2)[(Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC] as an efficient peptide-PEG transporter tool for carrying adenovirus vector into cells. (Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC was coupled with 6-maleimidohexanoic acid N-hydroxysuccinimide ester and the resulting 6-[(Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC-succinimido]hexanoic acid N-hydroxysuccinimide ester reacted with adenovirus. The modified adenovirus with the peptide-PEG hybrid exhibited high gene expression even in a CAR-negative cell line, DC2.4.     

Adenoviruses use lactoferrin as a bridge for CAR-independent binding to and infection of epithelial cells

Most adenoviruses bind to the coxsackie- and adenovirus receptor (CAR). Surprisingly, CAR is not expressed apically on polarized cells and is thus not easily available to viruses. Consequently, alternative mechanisms for entry of coxsackievirus and adenovirus into cells have been suggested. We have found that tear fluid promotes adenovirus infection, and we have identified human lactoferrin (HLf) as the tear fluid component responsible for this effect. HLf alone was found to promote binding of adenovirus to epithelial cells in a dose-dependent manner and also infection of epithelial cells by adenovirus. HLf was also found to promote gene delivery from an adenovirus-based vector. The mechanism takes place at the binding stage and functions independently of CAR. Thus, we have identified a novel binding mechanism whereby adenovirus hijacks HLf, a component of the innate immune system, and uses it as a bridge for attachment to host cells.     

Rhinovirus-mediated endosomal release of transfection complexes.

Endocytosis is an efficient method for transfer of genes into mammalian cells. Incorporation of adenovirus particles into gene transfer complexes greatly enhances gene delivery, probably by the release of endocytosed DNA into the cytoplasm. We report here that two different serotypes of human rhinovirus (HRV), HRV2 and HRV14, are also able to enhance receptor-mediated gene transfer. The effect of several compounds known to inhibit viral infection on HRV2- and HRV14-enhanced transfection was examined. WIN I(s) and WIN IV, two compounds which inhibit viral uncoating, had different effects on HRV2- and HRV14-enhanced gene transfer to NIH 3T3 cells. While HRV14-enhanced gene transfer was severely reduced in the presence of these compounds, virtually no effects were observed when HRV2 was used. The use of antiviral compounds thus allowed transfection of human cells, which are normally lysed rapidly upon infection with HRV. Viral activity could be mimicked by using a peptide derived from the N terminus of VP1 of HRV2. This peptide possesses pH-dependent membrane-disrupting activity and enhances gene transfer to NIH 3T3 and HeLa cells.     

Transferrin Receptor-Mediated Endocytosis: A Useful Target for Cancer Therapy

Current cancer management strategies fail to adequately treat malignancies with multivariable dose-restricting factors such as systemic toxicity and multi-drug resistance limiting therapeutic benefit, quality of life and complete long-term remission rates. The targeted delivery of a therapeutic compound aims to enhance its circulation and cellular uptake, decrease systemic toxicity and improve therapeutic benefit with disease specificity. The transferrin peptide, its receptor and their biological significance, has been widely characterised and vastly relevant when applied to targeting strategies. Utilising knowledge about the physiological function of the transferrin–transferrin receptor complex and the efficiency of its receptor-mediated endocytosis provides rationale to continue the development of transferrin-targeted anticancer modalities. Furthermore, multiple studies report an upregulation in expression of the transferrin receptor on metastatic and drug resistant tumours, highlighting its selectivity to cancer. Due to the increased expression of the transferrin receptor in brain glioma, the successful delivery of anticancer compounds to the tumour site and the ability to cross the blood brain barrier has shown to be an important discovery. Its significance in the development of cancer-specific therapies is shown to be important by direct conjugation and immunotoxin studies which use transferrin and anti-transferrin receptor antibodies as the targeting moiety. Such conjugates have demonstrated enhanced cellular uptake via transferrin-mediated mechanisms and increased selective cytotoxicity in a number of cancer cell lines and tumour xenograft animal models. In addition, incubation of chemotherapy-insensitive cancer cells with transferrin-targeted conjugates in vitro has resulted in a reversal of their drug resistance. Transferrin immunotoxins have also shown similar promise, with a diphtheria toxin mutant covalently bound to transferrin (Tf-CRM107) currently involved in human clinical trials for the treatment of glioblastoma. Despite this, the inability to translate preliminary research into a clinical setting has compelled research into novel targeting strategies including the use of nanoparticulate theory in the design of drug delivery systems. The main objective of this review is to evaluate the importance of the transferrin–transferrin receptor complex as a target for cancer therapy through extensive knowledge of both the physiological and pathological interactions between the complex and different cell types. In addition, this review serves as a summary to date of direct conjugation and immunotoxin studies, with an emphasis on transferrin as an important targeting moiety in the directed delivery of anticancer therapeutic compounds.     

A new peptide vector for efficient delivery of oligonucleotides into mammalian cells.

The development of antisense and gene therapy has focused mainly on improving methods for oligonucleotide and gene delivery into cells. In the present work, we describe a potent new strategy for oligonucleotide delivery based on the use of a short peptide vector, termed MPG (27 residues), which contains a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain derived from the nuclear localization sequence of SV40 T-antigen. The formation of peptide vector/oligonucleotide complexes was investigated by measuring changes in intrinsic tryptophan fluorescence of peptide and of mansyl-labelled oligonucleotides. MPG exhibits relatively high affinity for both single- and double-stranded DNA in a nanomolar range. Based on both intrinsic and extrinsic fluorescence titrations, it appears that the main binding between MPG and oligonucleotides occurs through electrostatic interactions, which involve the basic-residues of the peptide vector. Further peptide/peptide interactions also occur, leading to a higher MPG/oligonucleotide ratio (in the region of 20/1), which suggests that oligonucleotides are most likely coated with several molecules of MPG. Premixed complexes of peptide vector with single or double stranded oligonucleotides are delivered into cultured mammalian cells in less than 1 h with relatively high efficiency (90%). This new strategy of oligonucleotide delivery into cultured cells based on a peptide vector offers several advantages compared to other commonly used approaches of delivery including efficiency, stability and absence of cytotoxicity. The interaction with MPG strongly increases both the stability of the oligonucleotide to nuclease and crossing of the plasma membrane. The mechanism of cell delivery of oligonucleotides by MPG does not follow the endosomal pathway, which explains the rapid and efficient delivery of oligonucleotides in the nucleus. As such, we propose this peptide vector as a powerful tool for potential development in gene and antisense therapy.     

A synthetic peptide containing loop 4 of nerve growth factor for targeted gene delivery

BACKGROUND: Gene delivery vectors that restrict the expression of a therapeutic gene to a particular type of cells are critical to gene therapy in a complex structure, such as the central nervous system. We constructed a nonviral vector for targeted gene transfer to cells expressing nerve growth factor (NGF) receptor TrkA. METHODS AND RESULTS: The vector was a synthetic chimeric peptide composed of a targeting moiety derived from NGF loop 4 and a DNA-binding moiety of 10 lysine residues. The peptide activated signal transduction pathways of the NGF receptor TrkA in PC12 cells and supported the survival of the cells after serum deprivation. After forming complexes with plasmid DNA, the peptide dose-dependently increased reporter gene expression in PC12 cells, which could be inhibited by excess NGF. The peptide-mediated gene expression was not affected in PC12 cells by co-incubation with a blocking antibody against the low-affinity NGF receptor p75 and was significantly enhanced in NIH3T3 cells stably transfected with TrkA cDNA, suggesting the involvement of the high-affinity NGF receptor TrkA without the participation of p75. Moreover, the peptide did not assist gene transfer in TrkA-poor, but TrkB- and/or TrkC-positive primary cerebellar granule neurons and primary cortical glial cells. CONCLUSIONS: The chimeric peptide reported will be useful in gene delivery to and gene therapy of the nervous system and other tissues/organs with cells expressing TrkA.     

Improved gene delivery to human saphenous vein cells and tissue using a peptide-modified adenoviral vector

The establishment of efficient gene delivery to target human tissue is a major obstacle for transition of gene therapy from the pre-clinical phases to the clinic. The poor long-term patency rates for coronary artery bypass grafting (CABG) is a major clinical problem that lacks an effective and proven pharmacological intervention. Late vein graft failure occurs due to neointima formation and accelerated atherosclerosis. Since CABG allows a clinical window of opportunity to genetically modify vein ex vivo prior to grafting it represents an ideal opportunity to develop gene-based therapies. Adenoviral vectors have been frequently used for gene delivery to vein ex vivo and pre-clinical studies have shown effective blockade in neointima development by overexpression of candidate therapeutic genes. However, high titers of adenovirus are required to achieve sufficient gene delivery to provide therapeutic benefit. Improvement in the uptake of adenovirus into the vessel wall would therefore be of benefit. Here we determined the ability of an adenovirus serotype 5 vector genetically-engineered with the RGD-4C integrin targeting peptide inserted into the HI loop (Ad-RGD) to improve the transduction of human saphenous vein smooth muscle cells (HSVSMC), endothelial cells (HSVEC) and intact saphenous vein compared to a non-modified virus (Ad-CTL). We exposed each cell type to virus for 10, 30 or 60 mins and measured transgene at 24 h post infection. For both HSVSMC and HSVEC Ad-RGD mediated increased transduction, with the largest increases observed in HSVSMC. When the experiments were repeated with intact human saphenous vein (the ultimate clinical target for gene therapy), again Ad-RGD mediated higher levels of transduction, at all clinically relevant exposures times (10, 30 and 60 mins tissue:virus exposure). Our study demonstrates the ability of peptide-modified Ad vectors to improve transduction to human vein graft cells and tissue and has important implications for gene therapy for CABG.     

Mannose polyethylenimine conjugates for targeted DNA delivery into dendritic cells.

Cell surface-bound receptors represent suitable entry sites for gene delivery into cells by receptor-mediated endocytosis. Here we have taken advantage of the mannose receptor that is highly expressed on antigen-presenting dendritic cells for targeted gene transfer by employing mannosylpolyethylenimine (ManPEI) conjugates. Several ManPEI conjugates were synthesized and used for formation of ManPEI/DNA transfection complexes. Conjugates differed in the linker between mannose and polyethylenimine (PEI) and in the size of the PEI moiety. We demonstrate that ManPEI transfection is effective in delivering DNA into mannose receptor-expressing cells. Uptake of ManPEI/DNA complexes is receptor-specific, since DNA delivery can be competed with mannosylated albumin. Additionally, incorporation of adenovirus particles into transfection complexes effectively enhances transgene expression. This is particularly important for primary immunocompetent dendritic cells. It is demonstrated here that dendritic cells transfected with ManPEI/DNA complexes containing adenovirus particles are effective in activating T cells of T cell receptor transgenic mice in an antigen-specific fashion.     

Effect of transferrin as a ligand of pH-sensitive fusogenic liposome-lipoplex hybrid complexes

We previously developed potent nonviral vectors based on complexation of lipoplexes and pH-sensitive fusogenic liposomes, which achieve efficient transfection through membrane fusion with intracellular acidic compartments such as endosomes. Because transferrin receptor is known to be overexpressed in cancer cells, in this study, we investigated the effect of transferrin as a ligand for transfection of various cancer-derived cell lines mediated by the liposome-lipoplex hybrid complexes. Results showed that these hybrid complexes with transferrin exhibited higher transfection efficiency toward these cells than complexes without transferrin, but the extent of the transferrin-induced enhancement was dependent on the cell line. Conjugation of transferrin increased their transfection activity for HeLa and KB cells, although it only slightly enhanced transfection for HT1080, HepG2, and K562. Transferrin receptors in HT1080, HepG2, and K562 cells were internalized slowly, whereas those in HeLa and KB cells were internalized quickly and actively. These results indicate that transfection mediated by the ligand-attached hybrid complex does not correlate with the amount of transferrin receptor in the cell surface but correlate with the activity of internalization of transferrin receptor into the cells.     

Dynamin is required for recombinant adeno-associated virus type 2 infection

Recombinant adeno-associated virus (rAAV) vectors for gene therapy of inherited disorders have demonstrated considerable potential for molecular medicine. Recent identification of the viral receptor and coreceptors for AAV type 2 (AAV-2) has begun to explain why certain organs may demonstrate higher efficiencies of gene transfer with this vector. However, the mechanisms by which AAV-2 enters cells remain unknown. In the present report, we have examined whether the endocytic pathways of rAAV-2 are dependent on dynamin, a GTPase protein involved in clathrin-mediated internalization of receptors and their ligands from the plasma membrane. Using a recombinant adenovirus expressing a dominant-inhibitory form of dynamin I (K44A), we have demonstrated that rAAV-2 infection is partially dependent on dynamin function. Overexpression of mutant dynamin I significantly inhibited AAV-2 internalization and gene delivery, but not viral binding. Furthermore, colocalization of rAAV and transferrin in the same endosomal compartment provides additional evidence that clathrin-coated pits are the predominant pathway for endocytosis of AAV-2 in HeLa cells.     

Characterization of a cDNA encoding the bovine coxsackie and adenovirus receptor.

Non-human adenoviruses such as bovine adenovirus type 3 (BAV-3) that do not replicate in human cells but can infect human cells in culture could provide an attractive alternative to human adenoviral vectors for gene therapy. In addition, a large-animal model for genetic diseases can be very useful for the assessment of the efficacy of adenovector-mediated gene delivery in man. Recombinant human subgroup C adenovectors use the coxsackie and adenovirus receptor (CAR) to enter their target cells. Through RT-PCR and sequencing we determined the complete coding sequence of bovine CAR which serves as the primary adenoviral attachment site on bovine cells. A multiple sequence alignment, involving all the previously identified CAR species (man, mouse, rat, pig, and dog) showed that bovine CAR was most related to porcine CAR (92% nucleotide similarity) and demonstrated a highly conserved adenovirus binding Ig1 domain.     

Incorporation of Porcine Adenovirus 4 Fiber Protein Enhances Infectivity of Adenovirus Vector on Dendritic Cells: Implications for Immune-Mediated Cancer Therapy

One strategy in cancer immunotherapy is to capitalize on the key immunoregulatory and antigen presenting capabilities of dendritic cells (DCs). This approach is dependent on efficient delivery of tumor specific antigens to DCs, which subsequently induce an anti-tumor T-cell mediated immune response. Human adenovirus serotype 5 (HAdV5) has been used in human studies for gene delivery, but has limited infection in DCs, which lack the proper receptors. Addition of the porcine fiber knob (PK) from porcine adenovirus type 4 to HAdV5 allows the virus to deliver genetic material via binding to glycosylated surface proteins and bypasses the coxsackie-and-adenovirus receptor required by wild-type HAdV5. In this study we explored the potential therapeutic applications of an adenovirus with PK-based tropism against cancers expressing mesothelin. Infectivity and gene transfer assays were used to compare Ad5-PK to wild-type HAdV5. Mouse models were used to demonstrate peptide specificity and T-cell responses. We show that the PK modification highly augmented infection of DCs, including the CD141+ DC subset, a key subset for activation of naïve CD8+ T-cells. We also show that Ad5-PK increases DC infectivity and tumor specific antigen expression. Finally, vaccination of mice with the Ad5-PK vector resulted in enhanced T-cell-mediated interferon gamma (IFN-γ) release in response to both mesothelin peptide and a tumor line expressing mesothelin. Ad5-PK is a promising tool for cancer immunotherapy as it improves infectivity, gene transfer, protein expression, and subsequent T-cell activation in DCs compared to wild-type HAdV5 viruses.     

Transferrin-containing, cyclodextrin polymer-based particles for tumor-targeted gene delivery

Transferrin is a well-studied ligand for tumor targeting due to upregulation of transferrin receptors in numerous cancer cell types. Here, we report the development of a transferrin-modified, cyclodextrin polymer-based gene delivery system. The delivery system is comprised of a nanoparticle (formed by condensation of a cyclodextrin polycation with nucleic acid) that is surface-modified to display poly(ethylene glycol) (PEG) for increasing stability in biological fluids and transferrin for targeting of cancer cells that express transferrin receptor. A transferrin-PEG-adamantane conjugate is synthesized for nanoparticle modification. The transferrin conjugate retains high receptor binding and self-assembles with the nanoparticles by adamantane (host) and particle surface cyclodextrin (guest) inclusion complex formation. At low transferrin modification, the particles remain stable in physiologic salt concentrations and transfect K562 leukemia cells with increased efficiency over untargeted particles. The increase in transfection is eliminated when transfections are conducted in the presence of excess free transferrin. The transferrin-modified nanoparticles are appropriate for use in the systemic delivery of nucleic acid therapeutics for metastatic cancer applications.     

Preparation of a Tat-related transporter peptide for carrying the adenovirus vector into cells

We synthesized a Tat-related peptide acetyl-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-Gly-Cys amide, Ac-Tat(48-60)-Gly-Cys-NH(2), having high intracellular permeability, and conjugated this peptide to adenovirus vector to enhance gene transfer efficiency of adenovirus vector into cells. The peptide was prepared by the solid-phase peptide synthesis method and a bifunctional crosslinker 6-maleimidohexanoic acid N-hydroxysuccinimide ester was used to conjugate the peptide to adenovirus vector containing luciferase gene. The novel conjugate of adenovirus vector and Ac-Tat(48-60)-Gly-Cys-NH(2) peptide exhibited excellent gene transfer efficacy in B16BL6 cells.     

Engineering novel cell surface receptors for virus-mediated gene transfer.

The absence of viral receptors is a major barrier to efficient gene transfer in many cells. To overcome this barrier, we developed an artificial receptor based on expression of a novel sugar. We fed cells an unnatural monosaccharide, a modified mannosamine that replaced the acetyl group with a levulinate group (ManLev). ManLev was metabolized and incorporated into cell-surface glycoconjugates. The synthetic sugar decorated the cell surface with a unique ketone group that served as a foundation on which we built an adenovirus receptor by covalently binding biotin hydrazide to the ketone. The artificial receptor enhanced adenoviral vector binding and gene transfer to cells that are relatively resistant to adenovirus infection. These data are the first to suggest the feasibility of a strategy that improves the efficiency of gene transfer by using the biosynthetic machinery of the cell to engineer novel sugars on the cell surface.     

A novel peptide, THALWHT, for the targeting of human airway epithelia

Targeting gene vectors to human airway epithelial cells may help to overcome the current inefficiency of gene transfer as the major problem confronting cystic fibrosis gene therapy. To elucidate novel ligands targeting abundant, apically located receptors on airway epithelial cells, a phage display library was screened for peptides binding with high affinity to such cells. This screening yielded a selectively enriched amino acid sequence, Thr-His-Ala-Leu-Trp-His-Thr (THALWHT). Subsequent binding studies confirmed that THALWHT-displaying phages bound much stronger than phages displaying control peptides to human airway epithelial cells. In contrast, no significant binding differences were observed on a variety of non-airway-derived human cell lines suggesting selective binding of the THALWHT motif to airway epithelia. Confocal microscopy of such cells after exposure to labelled synthetic THALWHT peptide indicated that its binding is followed by specific internalisation via endocytosis. A synthetic peptide comprising a cyclic CTHALWHTC domain and a DNA binding moiety enabled efficient targeted gene delivery into human airway epithelial cells. Competition assays with free THALWHT peptide confirmed the specificity of gene delivery. Thus, the THALWHT motif may prove a useful targeting moiety for both non-viral and viral gene therapy vectors.     

Targeting Adenoviral Vectors by Using the Extracellular Domain of the Coxsackie-Adenovirus Receptor: Improved Potency via Trimerization

Adenovirus binds to mammalian cells via interaction of fiber with the coxsackie-adenovirus receptor (CAR). Redirecting adenoviral vectors to enter target cells via new receptors has the advantage of increasing the efficiency of gene delivery and reducing nonspecific transduction of untargeted tissues. In an attempt to reach this goal, we have produced bifunctional molecules with soluble CAR (sCAR), which is the extracellular domain of CAR fused to peptide-targeting ligands. Two peptide-targeting ligands have been evaluated: a cyclic RGD peptide (cRGD) and the receptor-binding domain of apolipoprotein E (ApoE). Human diploid fibroblasts (HDF) are poorly transduced by adenovirus due to a lack of CAR on the surface. Addition of the sCAR-cRGD or sCAR-ApoE targeting protein to adenovirus redirected binding to the appropriate receptor on HDF. However, a large excess of the monomeric protein was needed for maximal transduction, indicating a suboptimal interaction. To improve interaction of sCAR with the fiber knob, an isoleucine GCN4 trimerization domain was introduced, and trimerization was verified by cross-linking analysis. Trimerized sCAR proteins were significantly better at interacting with fiber and inhibiting binding to HeLa cells. Trimeric sCAR proteins containing cRGD and ApoE were more efficient at transducing HDF in vitro than the monomeric proteins. In addition, the trimerized sCAR protein without targeting ligands efficiently blocked liver gene transfer in normal C57BL/6 mice. However, addition of either ligand failed to retarget the liver in vivo. One explanation may be the large complex size, which serves to decrease the bioavailability of the trimeric sCAR-adenovirus complexes. In summary, we have demonstrated that trimerization of sCAR proteins can significantly improve the potency of this targeting approach in altering vector tropism in vitro and allow the efficient blocking of liver gene transfer in vivo.