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Photochemical Transfection: A New Technology for Light-Induced, Site-Directed Gene Delivery
Abstract
The development of methods for specific delivery of therapeutic genes into target tissues is an important issue for the further progress of in vivo gene therapy. In this article we report on a novel technology, named photochemical transfection, to use light to direct a precise delivery of therapeutic genes to a desired location. The technology makes use of photosensitizing compounds that localize mainly in the membranes of endosomes and lysosomes. On illumination these membrane structures will be destroyed, releasing endocytosed DNA into the cell cytosol. Using a green fluorescent protein gene as a model we show that illumination of photosensitizer-treated cells induces a substantial increase in the efficiency of transfection by DNA-poly-L-lysine complexes. Thus, in a human melanoma cell line the light treatment can increase the transfection efficiency more than 20-fold, reaching transfection levels of about 50% of the surviving cells. In this article various parameters of importance for the use of this technology are examined, and the potential use of the technology in gene therapy is discussed.
... Delivery of oligonucleotides to other organs than the liver is however still a major problem. PCI-induced release of an oligonucleotide from endosomes was demonstrated for the first time by Høgset et al. [159]. Since then several groups have shown that such release also leads to enhanced biological activity of many different types of oligonucleotides. ...
... In vitro, PCI gives a strong enhancement of plasmid delivery mediated by cationic polymers, with good effects shown with, e.g., cationic poly-amino acids [159], PEI [85,182] and PAMAM dendrimers [183]. While some such vehicles (e.g., PEI) have an inherent endosomolytic function, others, like poly-L-lysine, have no such properties [184] and with the latter type of vehicles PCI can essentially turn plasmid transfection on, against a background of no transfection. ...
... Polycations can be used for effecting nucleic acid uptake by conferring a positive charge to the negatively charged nucleic acids so that a polycation/nucleic acid complex can be taken up after electrostatic interaction with the negatively charged cell surface. This is a general uptake mechanism that works very well in combination with PCI with many types of nucleic acids ranging from PNAs conjugated to positively charged amino acids [163,217] thorough poly-amino acid mediated delivery of plasmids and mRNA [159,166], to adenovirus coated with polycations [208]. To the extent that the employed polycations are not able to induce a "proton sponge" effect, such systems will often have very low gene delivery efficiency on their own, meaning that PCI can be used to literally turn delivery on, with enhancement factors up to >100 times being observed [85,190]. ...
Photochemical internalisation (PCI) is a unique intervention which involves the release of endocytosed macromolecules into the cytoplasmic matrix. PCI is based on the use of photosensitizers placed in endocytic vesicles that, following light activation, lead to rupture of the endocytic vesicles and the release of the macromolecules into the cytoplasmic matrix. This technology has been shown to improve the biological activity of a number of macromolecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins (RIPs), gene-encoding plasmids, adenovirus and oligonucleotides and certain chemotherapeutics, such as bleomycin. This new intervention has also been found appealing for intracellular delivery of drugs incorporated into nanocarriers and for cancer vaccination. PCI is currently being evaluated in clinical trials. Data from the first-in-human phase I clinical trial as well as an update on the development of the PCI technology towards clinical practice is presented here.
... Together with the new fluorescence techniques on an in vivo rat bladder cancer model, the PCI technology was introduced on AY27 cells by PCI-induced gene transfection ( pEFGP plasmid complexes) to poly-lysine employing the photosensitizer TPPS 2a (LumiTrans®) as previously described. 36,37 In this study, it was desirable to document if AY27 cells, in general, are suitable for the PCI technology because the same cell line has earlier shown a successful bleomycin uptake by PCI. 19 ...
... As reported for other cell lines, enhanced transfection efficiency coincided with light-induced cell toxicity with transfection efficiency leveling at about 50% cell killing. 37 In TPCS 2a -treated AY27 cells, LD 50 doses with red light were about 2 times higher than those with blue light (Fig. 5A and B), a result in accordance with the results achieved for ALAbased PDT on AY27 cells. 45 A suitable TPCS 2a concentration of 0.2 µg ml −1 was administered in both in vitro and in vivo experiments on AY27 cells and was based on previous pilot studies. ...
... 50 Depending on the photosensitizer and actual light dose, rapid photobleaching may cause incomplete tumor destruction. 37,47 Further experiments are necessary to optimize irradiation with respect to the cell type, light intensity and exposure time and also to study effects by pulsed irradiation and other parameters. The bleaching rate of TPCS 2a is likely to indicate the one in tissue in vivo treatment, i.e. photosensitizer molecules will not bleach before the biological oxygen effect is achieved. ...
The possibility of using photochemical internalization (PCI) to enhance effects of the cytotoxic drug bleomycin is investigated, together with photophysical determinations and outlines of a possible treatment for intravesical therapy of bladder cancer. In vitro experiments indicated that employment of PCI technology using the novel photosensitizer TPCS2a® can enhance the cytotoxic effect of bleomycin in bladder cancer cells. Furthermore, experiments in an orthotopic in vivo bladder cancer model show an effective reduction in both necrotic area and bladder weight after TPCS2a based photodynamic therapy (PDT). The tumor selectivity and PDT effects may be sufficient to destroy tumors without damaging detrusor muscle layer. Our results present a possible new treatment strategy for non-muscle invasive bladder cancer, with intravesical instillation of photosensitizer and bleomycin followed by illumination through an optic fiber by using a catheter.
... Since endosomal escape is often an inefficient process, release of the transgene from endosomes represents one of the most important barriers for gene transfer. Photochemical internalization has been studied as a gene delivery technology (reviewed in Ref. 20) both with several nonviral212223 and with adenoviral vectors [24] as well as adeno-associated virus [25], mainly by using reporter genes such as genes encoding EGFP (enhanced green fluorescent protein) or ß-galactosidase. However, PCI-mediated gene delivery has also been shown to induce the delivery of therapeutic genes, such as the genes encoding HSV-tk (herpes simplex virus thymidine kinase) [26], p53 [27], PTEN [28], TRAIL [29], and IL-12 (interleukin-12) (unpublished results). ...
... The influence of PCI on transgene expression is illustrated inFigure 17.4. PCI has been shown to increase polycation transfection in some cases 100-fold, cationic lipid-based transfection fivefold, and adenoviral transduction 30-fold [21, 23, 24]. Adenovirus vectors are known to be taken into cells by endocytosis and to be released from endosomes by a regulated process. ...
IntroductionPhotodynamic TherapyPhotochemical Internalization (PCI)Conclusion
References
... Ponadto w SDT często wykorzystuje się te same sensybilizatory, co w PDT33343536, co znacznie ułatwia łączne stosowanie tych metod. W ostatnich latach niemałym zainteresowaniem badaczy również cieszy się nowatorska odmiana fotochemioterapii zwana internalizacją fotochemiczną (photochemical internalization -PCI), wykorzystująca powinowactwo niektórych sensybilizatorów do błon endosomów uczestniczących w endocytozie biologicznie aktywnych makrocząsteczek, których bezpośrednie przenikanie przez błony komórkowe jest niemożliwe373839404142. W metodzie tej farmakologicznie czynne makrocząsteczki przenoszone są do wnętrza komórki za pomocą endosomów, w których błonach inkorporowane są cząsteczki fotosensybilizatora. ...
... Co.) Metoda PCI pozwala na wprowadzanie do komórek i transport do receptorów wielu związków biologicznie czynnych podatnych na rozkład enzymatyczny, takich jak oligopeptydy, oligonukleotydy czy geny. Rozważa się zatem możliwość łączenia tej odmiany PDT z terapią genową lub immunoterapią [37, 38,404142. Pozwoliłoby to uniknąć niedogodności, związanych ze stosowaniem innych metod stanowiących połączenie PDT z immunoterapią, takich jak fotoimmunoterapia celowana (targeted photoimmunotherapy), w której fotosensybilizator w celu zwiększenia powinowactwa do tkanki nowotworowej wiązany jest z przeciwciałem monoklonalnym [13, 43]. ...
... In this study, we analyzed intracellular trafficking of non-cationic LPs by superresolution microscopy. Thereafter, in order to deliver siRNA using non-cationic LPs, we investigated the use of a photosensitizer that enables photochemical internalization (PCI) [9][10][11][12][13]. PCI is a method to facilitate the endosomal escape of reagents into cytoplasm using photosensitizer and light. ...
Small interfering RNA (siRNA) can specifically suppress gene expression by cleaving mRNA in the cytoplasm, termed RNA interference (RNAi). Although a nanoparticle-based siRNA delivery system has been approved for clinical use, the efficiency of cytoplasmic delivery of siRNA is still low. Recently, our group has established a highly efficient siRNA encapsulation method using non-cationic liposomes (LPs), which are more biocompatible than conventional cationic LPs. While non-cationic LPs containing siRNA were taken up by cells in vitro, the cytoplasmic release of siRNA and subsequent phenomena involving RNAi were not observed. It was considered that cytoplasmic delivery of siRNA, and siRNA release from LPs, should occur sequentially. We utilized amphiphilic photosensitizer Al (III) phthalocyanine chloride disulfonic acid (AlPcS2a), which can generate singlet oxygen under exposure to red light. When SK-HEP-1 cells expressing luciferase were treated with non-cationic LPs containing both siRNA and AlPcS2a, RNAi could be observed upon red light exposure, suggesting that singlet oxygen efficiently disrupts the membranous structures of endo/lysosomes and LPs. Thus, the combined use of AlPcS2a and red light is effective for utilizing non-cationic LPs for cytoplasmic delivery of siRNA.
... Besides these therapies, PS can also assist in the administration of other drugs, e.g., in gene therapy, through the so-called photochemical inter- nalization (PCI) technique. 15,16 PCI is an intracellular delivery method that increases the endosomal release of the drug by co-administration of PS agents, which help in disrupting the membranes of endocytic vesicles upon activation by light. On the other hand, if PS convert the absorbed light into a fluorescence emission, they can also be used for optical imaging. ...
The development of photoactive and biocompatible nanomaterials is a current major challenge of materials science and nanotechnology, as they will contribute to promoting current and future biomedical applications. A growing strategy in this direction consists of using biologically inspired hybrid materials to maintain or even enhance the optical properties of chromophores and fluorophores in biological media. Within this area, porphyrinoids constitute the most important family of organic photosensitizers. The following extensive review will cover their incorporation into different kinds of photosensitizing biohybrid materials, as a fundamental research effort toward the management of light for biomedical use, including technologies such as photochemical internalization (PCI), photoimmunotherapy (PIT), and theranostic combinations of fluorescence imaging and photodynamic therapy (PDT) or photodynamic inactivation (PDI) of microorganisms.
... A prominent method is PCI, which is achieved by disintegrating endosomal membranes through ROS produced by photosensitizers upon photoirradiation, facilitating the release of gene carriers into the cytosol. 271,410,411 As discussed in section 5.1, photosensitizers can be incorporated through polyion complexation into polymeric micelles. Thus, coloading photosensitizers and pDNA into the same structure allows photoinduced endosomal escape for improving transfection efficacy. ...
Polymeric micelles are demonstrating high potential as nanomedicines capable of controlling the distribution and function of loaded bioactive agents in the body, effectively overcoming biological barriers, and various formulations are engaged in intensive preclinical and clinical testing. This Review focuses on polymeric micelles assembled through multimolecular interactions between block copolymers and the loaded drugs, proteins, or nucleic acids as translationable nanomedicines. The aspects involved in the design of successful micellar carriers are described in detail on the basis of the type of polymer/payload interaction, as well as the interplay of micelles with the biological interface, emphasizing on the chemistry and engineering of the block copolymers. By shaping these features, polymeric micelles have been propitious for delivering a wide range of therapeutics through effective sensing of targets in the body and adjustment of their properties in response to particular stimuli, modulating the activity of the loaded drugs at the targeted sites, even at the subcellular level. Finally, the future perspectives and imminent challenges for polymeric micelles as nanomedicines are discussed, anticipating to spur further innovations.
... The cytotoxicity of different formulations of BLM against HeLa and MCF-7 cells was measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide blue-indicator dye]-based assay [16]. Cancer cells were cultured in medium supplemented with 10% FBS, 100 mg/mL streptomycin, 100 units/mL penicillin and 0.25 mg/mL amphotericin B, in a humidified atmosphere containing 5% CO 2 at 37 C. Approximately 1 Â 10 4 cells per well were seeded to a 96-well plate in 200 lL medium, and incubated for 24 h. ...
Targeted drug delivery has received considerable attention due to its key role in improving therapeutic efficacy and reducing the side effects of anticancer drugs. Bleomycin (BLM) is an anticancer antibiotic with short half-life, low therapeutic and high side effects that limit its clinical applications. This study aims to evaluate the anticancer potential of folate-targeted liposomal bleomycin (FL-BLM) and its free-folate form (L-BLM) on two different cancer cell lines including human cervix carcinoma HeLa, and human breast carcinoma MCF-7 cells. Furthermore, the effect of FL-BLM in induction of apoptosis and cell cycle arrest was studied by flow cytometry. FL-BLM was prepared by thin film hydration method and folic acid was conjugated to nanoliposomes by post insertion technique. Anticancer activity was evaluated by MTT assay. The cytotoxicity of FL-BLM against HeLa cells was significantly increased compared to L-BLM and conventional BLM. Flow cytometry and annexin-V analysis indicated that FL-BLM effectively induced apoptosis and cell-cycle arrest in HeLa cells especially at G2/M phase. In addition, the uptake of FL-BLM by Hela cells was significantly increased as compared to the MCF-7 cells. Overall, our findings indicated that FL-BLM may be promising formulation for targeted drug delivery to folate receptor-positive tumour cells.
... 17,18 Thus, extensive preclinical studies have indicated that photochemical internalisation could be a safe and highly specifi c anticancer treatment. 1,5,6,[10][11][12][13][14][15] By contrast with most other anticancer cytotoxic drugs, the chemotherapeutic bleomycin has some unusual physicochemical properties, including hydrophilicity and large size (1·4 kDa). These properties make bleomycin an agent that is largely taken up into cells by endocytosis, with accumulation in endocytic vesicles severely restricting its activity. ...
Background:
Photochemical internalisation, a novel minimally invasive treatment, has shown promising preclinical results in enhancing and site-directing the effect of anticancer drugs by illumination, which initiates localised chemotherapy release. We assessed the safety and tolerability of a newly developed photosensitiser, disulfonated tetraphenyl chlorin (TPCS2a), in mediating photochemical internalisation of bleomycin in patients with advanced and recurrent solid malignancies.
Methods:
In this phase 1, dose-escalation, first-in-man trial, we recruited patients (aged ≥18 to <85 years) with local recurrent, advanced, or metastatic cutaneous or subcutaneous malignancies who were clinically assessed as eligible for bleomycin chemotherapy from a single centre in the UK. Patients were given TPCS2a on day 0 by slow intravenous injection, followed by a fixed dose of 15 000 IU/m(2) bleomycin by intravenous infusion on day 4. After 3 h, the surface of the target tumour was illuminated with 652 nm laser light (fixed at 60 J/cm(2)). The TPCS2a starting dose was 0·25 mg/kg and was then escalated in successive dose cohorts of three patients (0·5, 1·0, and 1·5 mg/kg). The primary endpoints were safety and tolerability of TPCS2a; other co-primary endpoints were dose-limiting toxicity and maximum tolerated dose. The primary analysis was per protocol. This study is registered with ClinicalTrials.gov, number NCT00993512, and has been completed.
Findings:
Between Oct 3, 2009, and Jan 14, 2014, we recruited 22 patients into the trial. 12 patients completed the 3-month follow-up period. Adverse events related to photochemical internalisation were either local, resulting from the local inflammatory process, or systemic, mostly as a result of the skin-photosensitising effect of TPCS2a. The most common grade 3 or worse adverse events were unexpected higher transient pain response (grade 3) localised to the treatment site recorded in nine patients, and respiratory failure (grade 4) noted in two patients. One dose-limiting toxicity was reported in the 1·0 mg/kg cohort (skin photosensitivity [grade 2]). Dose-limiting toxicities were reported in two of three patients at a TPCS2a dose of 1·5 mg/kg (skin photosensitivity [grade 3] and wound infection [grade 3]); thus, the maximum tolerated dose of TPCS2a was 1·0 mg/kg. Administration of TPCS2a was found to be safe and tolerable by all patients. No deaths related to photochemical internalisation treatment occurred.
Interpretation:
TPCS2a-mediated photochemical internalisation of bleomycin is safe and tolerable. We identified TPCS2a 0·25 mg/kg as the recommended treatment dose for future trials.
Funding:
PCI Biotech.
... Une autre méthode enfin, utilise des ondes lumineuses. Il s'agit de stimuler par ces ondes, des composés photosensibles qui déstabilisent les membranes (Hogset et al. 2000). Cette méthode a déjà permis le franchissement membranaire par des complexes nanométriques (Nishiyama et al. 2005). ...
By focusing on drug administration within and to the central nervous system and notably on glioblastoma, the aim of the present PhD thesis work was to develop tools and evaluate the behavior of particulate nanocarriers as regards to biological barrier crossing. Three types of nanocarriers with sizes varying from 20 to 100 nm were evaluated: polysaccharide nanoparticles, PLGA nanoparticles, and lipid nanocapsules. The behavior of those objects concerning blood constituents allowed to define that coating with transferrine of PLGA nanoparticles or insertion of phospholipids or BSA within polysaccharide nanoparticles reduce their recognition by the reticulo-endothelial system and improve their plasma-resident time. Those surface modifications are also associated with a possibility of internalization in F98 glioma target cells essentially influenced by the lipid or polymeric nature or the carrier. Clear-cut evaluation of the cellular and subcellular behavior of LNC within F98 cells allowed to demonstrate that if the nature of the carrier is involved, notably considering the recruitment of cholesterol-dependant endocytic pathways, the size, correlated to amounts of surfactant provided, had also its significance. 20nm LNC are consequently the more apt to allow lysosome escape of transported drug and demonstrated higher pharmacological activities in inducing cell death through paclitaxel loading. Those results provide, therefore, original links between the subcellular behavior of nanocarrier and the bioavailability anticancer drugs. New potentialities of biological barrier crossing and size-dependent abilities were demonstrated. Those observations emphasize the interest of developing comparative studies in order to rationalize the use of a define nanocarrier for a given drug on a given target. Regarding new nanomedicines development, it demonstrates the importance to establish justification between biological behavior of nanocarriers and their therapeutic relevance.
... Enfin, la forte affinité de ces composés tétrapyrroliques pour les systèmes membranaires, associée à leur propriétés photochimiques, leur a récemment ouvert un nouveau champ d'application : la « Photo-Chemical Internalisation » (PCI). Cette technique est basée sur la rupture photo-induite des vésicules d'endocytose, libérant dans le cytosol les molécules endocytées [158][159][160][161][162]. De nombreux agents thérapeutiques, notamment ceux développés dans le cadre des thérapies géniques, sont en effet des macromolécules (oligonucléotides, PNA, gènes, protéines, peptides…) qui ne peuvent pénétrer dans les cellules que par endocytose. ...
Thanks to their preferential retention in proliferating tissues, some photosensitizers are therapeutically used such as in photodynamic therapy (PDT). In most cases, they are based on the porphyrin structure, but other compounds, of which far-red-light absorption properties are most compatibles with biological tissues irradiation, have been developed. In this work, the focus is given on two amphiphilic tetrapyrroles: deuteroporphyrin (DP) that bears two carboxylic groups on one side of the macrocycle and disulfonated aluminum-phthalocyanine (AlPcS2a) that bears two sulfonated groups on one side. Indeed, the selectivity of photosenzitizers for cells in proliferation, as well as their intracellular localization, depends on their structure, in particular on their hydrophobicity and the symmetry of distribution of their polar chains around the macrocycle. Two major processes are involved. On one hand, lipophilic or amphiphilic photosensitizers possess a high affinity for low-density lipoproteins (LDL). The increased cholesterol catabolism of proliferating tissues results in over-expression of LDL receptors. Hence, LDL could act as natural carriers of photosensitizers and insure their targeting to tumor cells. On the other hand, the relative acid pH of extracellular medium could play an important role by governing the physico-chemical properties of photosensitizers and consequently their ability to cross membranes. In fine, the photosensitizers behavior seems to involve both the physicochemical and biological properties of the microenvironment.
In order to elucidate the implied mechanisms, we studied the interactions of photosensitizers with LDL and with liposomes (SUV, used as membranes-models) with a special interest on dynamics of these processes. A particular attention has been paid to the effects of pH. The data obtained on these simple systems then allowed us to interpret the sub-cellular localization of the photosensitizers on a human line of fibroblasts, and to evaluate the influence of LDL on the intracellular distribution of the compounds. This last point is of major importance because the localization of such photosensitizers (in particular AlPcS2a) in endocytic vesicles and their subsequent ability to induce a release of the contents of these vesicles - including externally added macromolecules - into the cytosol is the basis for a recent method for macromolecule activation, named photochemical internalization (PCI).
... PCI might not only enhance anticancer efficacy of therapeutic macromolecules but also decrease the development of drug-related side effects to the illuminated tissues. Moreover, the PCI strategy might serve as a mechanism of delivery of plasmids or adenoviruses for the purpose of gene therapy (124)(125). The most desirable photosensitizers to be used in the PCI are amphyphylic disulphonated compounds that localize to the vesicle membranes or tetrasulphonated ones that localize to the matrix of the endocytic vesicles (126). ...
Photodynamic therapy (PDT) is a clinically approved method of tumor treatment. Its unique mechanism of action results from minimal invasiveness and high selectivity towards transformed cells. However, visible light used to excite most photosensitizers has rather limited ability to penetrate tissues resulting in insufficient destruction of deeply seated malignant cells. Therefore, novel strategies for further potentiation of the anticancer effectiveness of PDT have been developed. These include combined treatments with surgery, chemo-and radiotherapy, strategies targeting cytoprotective mechanisms induced in PDT-treated cells, as well as attempts aimed at enhancement of PDT-mediated antitumor immune response. Moreover, new photosensitizers and novel light sources are being developed. Impressive progress in nanotechnology and understanding of tumor cell biology rise hopes for further improvements in this elegant and promising method of cancer treatment.
... Recent studies have shown encouraging results in gene transfection via either viral or nonviral vectors. AlPcS 2a -based PCI-induced EGFP/polylysine complex delivery was tested and the transfection efficiency reached above 50% of surviving cells [43]. PCI using different photosensitizers has also been examined to induce EGFP transfection, and a light-dependent transfect efficacy was found [10]. ...
Photodynamic therapy (PDT) is considered to be a site-specific cancer therapy because the therapeutic effects only take place in the area exposed to light. With the rapid expansion of PDT, many new drugs either chemically or endogenously synthesized have been developed and exploited in various ways. Among them, there is a group of photosensitizers with a unique intracellular distribution pattern observed under fluorescence microscopy. These photosensitizers enter cells through endocytosis and localize at the membrane of endocytic vesicles instead of being diffusely localized in the cytosol. Upon irradiation, the generated cytotoxic agents destroy the endocytic membranes and release the content inside these organelles (e.g., lysosomal enzymes) [1,2]. Moreover, the redistribution of these sensitizers (i.e., an increase of fluorescence intensity and the change of distribution phenomenon to become diffuse) was also observed. This particular lysosomal photosensitization and the membrane disruption accompanying the release of content led to the concept of photochemical internalization (PCI)—a novel photochemical technology for inducing the release of macromolecules from endocytic vesicles [3]. Macromolecules generally refer to oligo-and polynucleotides, peptides, proteins, and polymers whose sizes are normally larger than 1 kDa. They can serve as alternatives to traditional drugs (e.g., low-molecular-weight chemotherapeutics) but their bioavailabilities are usually limited [4]. First of all, the cell membrane is normally impermeable to the aforementioned large molecular complexes, whereas small molecules such as glucose, ions, or amino acids can quickly diffuse into cells. Secondly, even
... The first one has been developed for promoting cellular internalization, for example, by developing a polymer as a transfection agent of the active substance. This polymer contains an oxygen photosensitizer, [9][10][11] which upon irradiation produces reactive oxygen species that disrupt the endosomal membrane, thereby releasing the active compound into the cytosol. The second type of strategy, called "photo-caging", uses light to activate the ASO or siRNA by preventing unspecific reactions in the absence of illumination and thus releasing the drug only under irradiation. ...
A photoreactive ruthenium(II) complex that contains two tetraazaphenanthrene (TAP) and one phenanthroline (phen) ligands was synthesized and then tethered to (antisense) oligonucleotides (Ru–ASO) to target a destabilized GFP (dGFP). The specificity of the photoreaction of this Ru–ASO conjugate was studied in vitro by polyacrylamide gel electrophoresis (PAGE) experiments in denaturing conditions. Other nonspecific Ru–ASO conjugates were also prepared and evaluated with human keratinocytes that expressed dGFP. An illumination-dependent cytotoxicity was observed for most Ru–ASO conjugates that varied from 10 to almost 40 %, but only the specific Ru–ASO conjugate was able to significantly reduce GFP expression in illuminated cells.
... For evaluation of the ability of the nanocarriers to induce PCI, experiments were performed to investigate if the nanocarriers could enhance gene transfection with a plasmid/poly lysine complex. 1,46 The plasmid employed encodes enhanced green fluorescent protein (EGFP), and the expression of EGFP was analyzed by flow cytometry as described (see Experimental Section). Figure 7 shows the result of an experiment with the nanocarrier 16 (batch B) and indicates that this carrier compound induced at substantial enhancement of transfection upon illumination (an increase in % EGFP-positive cells from 0.2% with no illumination to 20% with 120 s illumination). ...
Water soluble amphiphilic chitosan nanocarriers tethered with the photosensitizer meso-tetraphenylporphyrin were synthesized, in a seven step procedure, starting from 3,6-di-O-tert-butyldimethylsilyl-chitosan and 5-(p-aminophenyl)-10,15,20-triphenylporphyrin. The lipophilic photosensitizer could be introduced in a quantitative and reproducible reaction to give either 0.1 or 0.25 degree of substitution per glucosamine monomer. Fluorescence and NMR investigations revealed the dynamic structures of the carriers, which formed nanoparticles in aqueous solution with a core of -stacked photosensitizers. These carriers can then unfold in the lipophilic environment and the photosensitizer moiety can thus be inserted into the cell membrane. The efficacy of the carriers for photochemical internalization (PCI) mediated gene delivery was evaluated in vitro using the HCT116/LUC human colon carcinoma cell line. The efficacy of transfection was comparable to what could be achieved by the reference compound and current clinical candidate TPCS2a.
... With most gene delivery systems the therapeutic gene is taken into the cell by endocytosis, and for many of these systems, especially non-virusbased ones, the lack of efficient mechanisms for translocating the gene out of the endocytic vesicles constitutes a major hindrance for realization of the therapeutic potential of the gene. PCI has been studied as a gene delivery technology (reviewed in [31] both with several non-viral323334 and viral vectors [35] mainly by using reporter genes such as genes encoding eGFP (enhanced green fluorescent protein) or ß-galactosidase. However, PCI-mediated gene delivery has also been shown to induce the delivery of therapeutic genes, such as the genes encoding HSV-tk (Herpes Simplex Virus thymidine kinase) [36] p53 [37], TRAIL ([38] unpublished results) and IL-12 (interleu- kin-12) (unpublished results). ...
The utilization of macromolecules in the therapy of cancer and other diseases is becoming increasingly important. Recent advances in molecular biology and biotechnology have made it possible to improve the targeting and design of cytotoxic agents, DNA complexes and other macromolecules for clinical applications. In most cases the targets of macromolecular therapeutics are intracellular. However, degradation of macromolecules in endocytic vesicles after uptake by endocytosis is a major intracellular barrier for the therapeutic application of macromolecules having intracellular targets of action.
... PCI has in vitro been shown in more than 80 cell lines to increase the biological activity of a large variety of clinically relevant drugs that do not readily penetrate the plasma membrane [4]. Examples are type I ribosome-inactivating proteins (RIPs) [5,6], immunotoxins789, gene-encoding plasmids [10,11] , ade- novirus121314, peptides [15], PNAs [16,17], and some chemotherapeutics such as bleomycin [18], doxorubicin [19,20], and mitoxantrone [21]. The PCI principle has also been demonstrated in vivo with the protein toxin gelonin [22], the p53 gene encoded on a plasmid [23], bleomycin [18] and recently also with the targeted protein toxin scFvMEL/rGel [24] . ...
Photochemical internalization (PCI) is a method for intracellular delivery of hydrophilic macromolecular drugs with intracellular targets as well as other drugs with limited ability to penetrate cellular membranes. Such drugs enter cells by means of endocytosis and are to a large extent degraded by hydrolytic enzymes in the lysosomes unless they possess a mechanism for cytosolic translocation. PCI is based on photodynamic therapy (PDT) specifically targeting the endosomes and lysosomes of the cells, so that the drugs in these vesicles can escape into the cytosol from where they can reach their targets. The preferential retention of the photosensitizer (PS) in tumor tissue in combination with controlled light delivery makes PCI relatively selective for cancer tissue. The tumor specificity of PCI can be further increased by delivery of drugs that selectively target the tumors. Indeed, this has been shown by PCI delivery of several targeted protein toxins. Targeted protein toxins may be regarded as ideal drugs for PCI delivery, and may represent the clinical future for the PCI technology.
... PCI might not only enhance anticancer efficacy of therapeutic macromolecules but also decrease the development of drug-related side effects to the illuminated tissues. Moreover, the PCI strategy might serve as a mechanism of delivery of plasmids or adenoviruses for the purpose of gene therapy (124)(125). The most desirable photosensitizers to be used in the PCI are amphyphylic disulphonated compounds that localize to the vesicle membranes or tetrasulphonated ones that localize to the matrix of the endocytic vesicles (126). ...
Photodynamic therapy (PDT) is a clinically approved method of tumor treatment. Its unique mechanism of action results from minimal invasiveness and high selectivity towards transformed cells. However, visible light used to excite most photosensitizers has rather limited ability to penetrate tissues resulting in insufficient destruction of deeply seated malignant cells. Therefore, novel strategies for further potentiation of the anticancer effectiveness of PDT have been developed. These include combined treatments with surgery, chemo- and radiotherapy, strategies targeting cytoprotective mechanisms induced in PDT-treated cells, as well as attempts aimed at enhancement of PDT-mediated antitumor immune response. Moreover, new photosensitizers and novel light sources are being developed. Impressive progress in nanotechnology and understanding of tumor cell biology rise hopes for further improvements in this elegant and promising method of cancer treatment.
mRNA therapy has shown great potential in infectious disease vaccines, cancer immunotherapy, protein replacement therapy, gene editing, and other fields due to its central role in all life processes. However, mRNA is challenging to pass through the cell membrane due to its significant negative charges and degradation from RNase, so the key to mRNA therapy is efficient packaging and delivery of it with appropriate vectors. Presently researchers have developed various vectors such as viruses and liposomes, but these conventional vectors are now difficult to meet the growing requirement like safety, efficiency, and targeting, so many novel delivery vectors with unique advantages have emerged recently. This review mainly introduces two categories of novel vectors: biomacromolecules and inorganic nanoparticles, as well as two novel methods of control and administration based on these novel vectors: controlled‐release administration and non‐invasive administration. These novel delivery strategies have the advantages of high safety, biocompatibility, versatility, intelligence, and targeting. This paper analyzes the challenges faced by the field of mRNA delivery in depth, and discusses how to use the characteristics of novel vectors and administrations to solve these problems.
Minimally invasive techniques such as Photodynamic Therapy (PDT) and Photochemical Internalisation (PCI) have for years been under investigation for the treatment of solid cancers. A significant number of the recent research studies have applied PDT and PCI to biological three-dimensional (3D) cancer platforms with many of the studies also involving the use of nanoparticles in order to enhance the efficacy of these light-based therapies. Interest in the employment of 3D cancer platforms has increased considerably due to the ability of the platforms to mimic in vivo models better than the conventional two-dimensional (2D) cultures. Some of the advantages of the 3D cancer systems over their 2D counterparts include improved interaction between cancer cells and the surrounding extracellular matrix (ECM) as well as restricted drug penetration which would allow optimization of treatments prior to undertaking of in vivo studies. The different chapters of this book will discuss photosensitizers and nanoparticles used in PDT and PCI in addition to the applications of these treatments in various 3D cancer models.
Cell sheets play a vital role in gene therapies. Nonetheless, gene delivery platforms based on cell sheet technology are restricted to small genes. Here, we produced a rapid and efficient light activated gene delivery nanodot platform for large genes. Due to its large gene sequence, low-density lipoprotein receptor-related protein 5 (LRP5) is rarely used for gene therapy in vivo. Our study successfully constructed a nanodot platform using light and adenoviruses to harvest LRP5-overexpressing bone marrow mesenchymal stem cell (BMSC) sheets with a nearly 2-fold increase in efficiency and a 40% reduction in time compared with a traditional gene delivery platform. Overexpression of LRP5 upregulated the expression levels of alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP2), and runt-related transcription factor-2 (Runx2). Further, in vivo results suggested that LRP5 enhanced BMSC sheet-mediated osseointegration. This light activated gene delivery nanodot platform may serve as a potentially powerful tool for introducing various genes and for increasing the scope of application for cell sheet-based gene therapies.
Background: Photochemical internalisation (PCI) is a light-triggered and site-specific technique that enhances the delivery of therapeutic agents to their intracellular targets using amphiphilic, photosensitizing agents. / Methods: This study investigated the effect that the intracellular redox environment of 4T1 breast cancer cells exerts on PCI-facilitated delivery of the type I ribosome inactivating protein, saporin, and the topoisomerase inhibitor, mitoxantrone, either individually or in combination. Buthionine sulfoximime (BSO), a clinically used inhibitor of glutathione synthesis, and the singlet oxygen scavenger, l-histidine, were used to enhance the oxidative and reductive state of the cells respectively. / Results: PCI of saporin at 30 nM was effective in reducing cellular viability, which decreased to 16% compared to "dark" controls (P < 0.01). Addition of BSO enhanced PCI efficacy by a further factor of three (P < 0.01), but addition of l-histidine completely inhibited cytotoxicity induced by PCI. The combination of the two cytotoxic agents, saporin and mitoxantrone, with PCI, elicited 14% and 17% reduction in cell viability (P < 0.01) compared to PCI with saporin alone and mitoxantrone alone respectively. Combination treatment with BSO resulted in a further significant reduction in cell viability by 18% (P < 0.01). / Conclusions: Our findings show the efficacy of PCI can be manipulated and potentiated by modifying the intracellular redox environment.
Electrostatics‐mediated self‐assembly formed from plasmid DNA (pDNA) and block catiomers composed of a neutral block and a cationic block has been gaining attention as a potential systemic gene delivery system. The mission of thus formed polyplex micelles is to deliver pDNA encoding therapeutic gene into the nucleus of targeted cells and to exert transgene expression. The key issues are controlled packaging of pDNA into polyplex micelles to suitably regulate their property and rational integration of necessary functionalities to accommodate a series of inherent physiological barriers in delivery. This chapter first focuses on unveiling the molecular mechanism of pDNA packaging followed by exploration of suitable structures and required functionalities to overcome each of the delivery process. Finally, molecular designs to manage entire systemic delivery process are outlined.
Interactions between stromal cells and tumor cells pay a major role in cancer growth and progression. This is reflected in the composition of anticancer drugs which includes compounds directed towards the immune system and tumor-vasculature in addition to drugs aimed at the cancer cells themselves. Drug-based treatment regimens are currently designed to include compounds targeting the tumor stroma in addition to the cancer cells. Treatment limiting adverse effects remains, however, one of the major challenges for drug-based therapy and novel tolerable treatment modalities with diverse high efficacy on both tumor cells and stroma is therefore of high interest. It was hypothesized that the vascular targeted fusion toxin VEGF121/rGel in combination with the intracellular drug delivery technology photochemical internalization (PCI) stimulate direct cancer parenchymal cell death in addition to inhibition of tumor perfusion, and that an immune mediated response is relevant for treatment outcome. The aim of the present study was therefore to elucidate the anticancer mechanisms of VEGF121/rGel-PCI. In contrast to VEGF121/rGel monotherapy, VEGF121/rGel-PCI was found to mediate its effect through VEGFR1 and VEGFR2, and a targeted treatment effect was shown on two VEGFR1 expressing cancer cell lines. A cancer parenchymal treatment effect was further indicated on H&E stains of CT26-CL25 and 4 T1 tumors. VEGF121/rGel-PCI was shown, by dynamic contrast enhanced MRI, to induce a sustained inhibition of tumor perfusion in both tumor models. A 50% complete remission (CR) of CT26.CL25 colon carcinoma allografts was found in immunocompetent mice while no CR was detected in CT26.CL25 bearing athymic mice. In conclusion, the present report indicate VEGF121/rGel -PCI as a treatment modality with multimodal tumor targeted efficacy that should be further developed towards clinical utilization.
Most nonviral gene transfection vectors deliver transfecting DNA into cells through the endocytic pathway (1,2). Poor escape from endocytic vesicles in many cases constitutes a major barrier for delivery of a functional gene, since the endocytosed transfecting DNA is unable to reach the cytosol and be further transported to the nucleus, but rather is trapped in endocytic vesicles and finally degraded in lysosomes (3). Therefore, the development of endosome-disruptive strategies is of great importance for the further progress of gene transfection. We have developed a new technology, termed photochemical internalization (PCI), to achieve light-inducible permeabilization of endocytic vesicles (4–8). The technology is based on photochemical reactions initiated by photosensitizers localized in endocytic vesicles and inducing rupture of these vesicles upon light exposure (4). This leads to the release of endocytosed macromolecules such as transfecting DNA from endocytic vesicles into the cytosol (Fig. 1). As a light-dependent treatment, PCI-mediated transfection (photochemical transfection) allows the possibility of directing the gene delivery to a desired site, e.g., achieving tumor-specific expression of a therapeutic gene in gene therapy in vivo. Fig. 1. Principle of photochemical internalization (PCI). I, endocytosis of the pho-tosensitizer (S) and the transfecting gene (G); II, localization of the photosensitizer and the transgene in the same endocytic vesicles; III, rupture of endosomal membrane upon light exposure and subsequent release of the transfecting gene into the cytosol.
Gene therapy has been considered to be a powerful approach for the prevention and/or treatment of a variety of diseases from genetic disorders, infections, to cancer. The success of gene therapy in the clinic is largely limited currently, mainly due to the lack of safe and efficient delivery vectors. Despite the high transfection efficiency, viral vectors encounter the vital toxicity issues and production problems. Increasing endeavors have been therefore directed towards the development of non-viral systems with the advantages of low immunogenicity and toxicity, ease in manufacturing and mass production, low cost, excellent stability, reduced vector size limitations, high flexibility regarding the size of transgenes to be delivered, and diverse chemical designs for constructing vectors with multiple functions. In this chapter, we summarized most of the synthetic non-viral systems currently employed for gene therapy, including lipid and polymer-based vectors, nanomaterials such as magnetic nanoparticles, quantum dots, gold/silica nanostructures, carbon nanotubes, calcium phosphate nanoparticles, and layered double hydroxides/clays, as well as multifunctional nanosystems among them. Particular attention has been paid on synthetic polymers and the related nanomedicines. Selected clinical trials of gene therapy using non-viral vectors as well as the future development in this rapidly growing field were briefly discussed.
Despite recent progresses in nanoparticle-based drug delivery systems, there are still many unsolved limitations. Most of all, a major obstacle in current nanoparticle-based drug carrier is the lack of sufficient drug delivery into target cells due to various biological barriers, such as: extracellular matrix, endolysosomal barrier, and drug-resistance associated proteins. To circumvent these limitations, several research groups have utilized photochemical internalization (PCI), an extension of photodynamic therapy (PDT), in design of innovative and efficient nano-carriers drug delivery. This review presents an overview of a recent research on utilization of PCI in various fields including: anti-cancer therapy, protein delivery, and tissue engineering.
For the last five decades cationic polymers have been used for nucleic acids transfection. Our understanding of polymer-nucleic acid interactions and their rational use in delivery has continuously increased. The great improvements in macromolecular chemistry and the recognition of distinct biological extra- and intracellular delivery hurdles triggered several breakthrough developments, including the discovery of natural and synthetic polycations for compaction of nucleic acids into stable nanoparticles termed polyplexes; the incorporation of targeting ligands and surface-shielding of polyplexes to enable receptor-mediated gene delivery into defined target tissues; and strongly improved intracellular transfer efficacy by better endosomal escape of vesicle-trapped polyplexes into the cytosol. These experiences triggered the development of second-generation polymers with more dynamic properties, such as endosomal pH-responsive release mechanisms, or biodegradable units for improved biocompatibility and intracellular release of the nucleic acid pay load. Despite a better biological understanding, significant challenges such as efficient nuclear delivery and persistence of gene expression persist. The therapeutic perspectives widened from pDNA-based gene therapy to application of novel therapeutic nucleic acids including mRNA, siRNA, and microRNA. The finding that different therapeutic pay loads require different tailor-made carriers complicates preclinical developments. Convincing evidence of medical efficacy still remains to be demonstrated. Bioinspired multifunctional polyplexes resembling "synthetic viruses" appear as attractive opportunity, but provide additional challenges: how to identify optimum combinations of functional delivery units, and how to prepare such polyplexes reproducibly in precise form? Design of sequence-defined polymers, screening of combinatorial polymer and polyplex libraries are tools for further chemical evolution of polyplexes.
This study demonstrates a strategy to enhance gene delivery via photoregulated gene unpacking from its vector. Photoresponsive polycationic vectors composed of a middle azobenzene moiety and two terminal blocks of poly[2-(dimethylamino)ethyl methacrylate], termed as Azo-PDMAEMA, are synthesized using a difunctional azobenzene-based initiator via atomic transfer radiator polymerization (ATRP). The Azo-PDMAEMA exhibits trans to cis isomerization under alternate Vis-UV irradiation, and is capable of condensing plasmid DNA into nanocomplexes. Hydrophobic azobenzene groups in the cationic polymers are shown to enhance the interaction of complexes with the cell membrane, thus improving cell uptake and transfection efficiency. Increased gene expression in COS-7 cells, HepG-2 cells and CHO-K1 cells is achieved after UV irradiation due to UV-triggered intracellular gene unpacking. Time-resolved fluorescence assays further indicate that the trans to cis photoisomerization of Azo-PDMAEMA induces less compacted complexes, contributing to more exposure of genes for transcription.
According to recent advances in nanotechnology, various nano-sized formulations have been designed for the application in biomedical fields, including diagnosis, drug delivery, and therapeutics. The nanotechnology-based formulations have a great merit in the design of multifunctional platform for the biomedical applications. Therefore, recent trends in nanotechnology are moving onto the combination of nanotechnology and conventional therapeutic. Typically, photodynamic therapy (PDT) is one of promising techniques for the combination with nanotechnology owing to its less invasiveness. In this paper, we are going to briefly review recent advances in nanotechnology-based PDT, including selective delivery and excitation of photosensitizers, combination therapy, and multifunctional PDT.
Photochemical internalization (PCI) is a method for releasing macromolecules from endosomal and lysosomal compartments. The PCI approach uses a photosensitizer that localizes to endosomal and lysosomal compartments, and a light source with appropriate light spectra for excitation of the photosensitizer. Upon photosensitizer excitation, endosomal and lysosomal membranes are destroyed, due to the formation of reactive oxygen species, followed by release of the endocytosed material. PCI has been demonstrated to enhance and control (site- and time-specific) delivery of various macromolecules such as viruses, proteins, chemotherapeutics, nucleic acid, and so on. In this Review we present past and current studies of PCI-controlled delivery of natural and artificial nucleic acids, such as peptide nucleic acids, siRNA molecules, mRNA molecules and plasmids. We also discuss critical aspects to further the possibilities for successful gene targeting in space and time.
Targeting cancer vasculature is an emerging field in cancer treatment. Photochemical internalization (PCI) is a drug delivery technology based on photochemical lysis of drug-bearing endocytic vesicles originally designed to target cancer cells. Recent investigations have revealed a lower PCI efficacy in vascular endothelial cells (HUVECs) in vitro than in HT1080 fibrosarcoma cells. This manuscript aims to explore the limiting factor for the PCI effect in HUVECs. Cellular uptake of the photosensitizers AlPcS2a and TPPS2a, and a model compound for macromolecular drugs taken up by fluid phase endocytosis, Alexa(488)-dextran, was explored by flow cytometry. The uptake of AlPcS2a and TPPS2a was 3.8-fold and 37-fold higher in HUVECs than in HT1080 cells, respectively, while the Alexa(488)-dextran uptake was 50% lower. AlPcS2a (but not TPPS2a) was shown to reduce Alexa(488)-dextran uptake in a concentration-dependent manner, resulting in 66% and 33% attenuation of Alexa(488)-dextran uptake at 20μg/ml AlPcS2a in HUVECs and HT1080 cells respectively. Studies of intracellular localization of Alexa(488)-dextran and AlPcS2a by confocal microscopy in HUVECs uncovered a concentration-dependent AlPcS2a-induced inhibition of Alexa(488)-dextran trafficking into AlPcS2a-stained and acidic vesicles. The localization of Alexa(488)-dextran to AlPcS2a-localizing compartments was reduced by 40% when the AlPcS2a concentration was increased from 5 to 20μg/ml. The treatment dose of AlPcS2a was found to influence on the efficacy of PCI of saporin, but to a lesser extent than expected considering the data from cellular uptake and intracellular trafficking of Alexa(488)-dextran. The implications of these results for further development of vascular targeting-PCI are discussed.
Recently, biomedical applications of dendrimers, three-dimensional tree-like macromolecules, have received much attention. In this paper, we introduce new biomedical applications of functional dendrimers: dendrimers encapsulating photosensitizers composed of a center dye molecule surrounded by poly(benzyl ether) dendrons with ionic peripheral groups as a new type of photosensitizer (PS) for photodynamic therapy (PDT). Dendrimers encapsulating porphyrin and with ionic peripheral groups (DPs) spontaneously formed polyion complex (PIC) micelles through electrostatic interactions with oppositely-charged block copolymers, and showed no self-quenching of the center dye molecule inside the micellar core due to a unique DP structure, leading to remarkable in vitro photocytotoxicity. The DP-incorporated micelles showed successful treatment of choroidal neovascularization (CNV) in rats without any sign of side effects. Thus, the DP-incorporated micelles are expected to be an innovative PS formulation for successful PDT against ophthalmic diseases. In this paper, we also review polymeric micelles incorporating phthalocyanine core dendrimers (DPc) for cancer PDT and novel light-responsive supramolecular gene carriers integrated with DPc for site-directed transfection in vivo.
We report a novel strategy to immobilize SDS-coated proteins for the fully integrated microfluidic Western blotting. Polyacrylamide gel conjugated with a cationic polymer, poly-L-lysine, effectively immobilizes all sized proteins after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and enables to integrate the SDS-PAGE and subsequent immuno-probing in an automated microfluidic chip. Through rational manipulation of the controllable poly-L-lysine for a charge-patterned matrix, immobilization strength can be easily optimized for the target analyte and detailed study of immobilization mechanism can be presented. The dependence of protein capture behavior on both the concentration of copolymerized charges and poly-lysine length is studied and gives important insight into the electrostatic immobilization mechanism. Based on the fluorescence analysis of immobilized proteins, the bound proteins refold and ready for successive immunoassay during electrophoretic removal of SDS. The automated on-chip Western blot for model proteins or a cell lysate is demonstrated step-by-step including injection, separation, transfer, immobilization, blocking, and immunoblot. The approach advances microfluidic protein immunoblotting which relates directly to the conventional SDS-PAGE based slab-gel western blot, while saving sample volume, labor and assay time.
Polymeric micelles, supramolecular assemblies of block copolymers, are useful nanocarriers for the systemic delivery of drugs and genes. Recently, novel polymeric micelles with smart functions, such as targetability and stimuli-sensitivity, have emerged as promising carriers that enhance the efficacy of drugs and genes with minimal side effects. This review focuses on the construction and characteristic behaviors of intracellular environment-sensitive micelles that selectively exert drug activity and gene expression in live cells.
A promising strategy for increased intracellular delivery of nucleic acids with the benefit for targeting is photochemical internalization (PCI). PCI relies on the use of a photosensitizing compound that photochemically destroys membranes in the endocytic pathway after illumination, resulting in cytosolic transfer of endosomal content. PCI technology combined with biodegradable polyamino acid carriers and nucleic acids delivers effective targeting and improved biosafety. In an in vitro model system, we have evaluated various poly-l-lysine (PLL), poly-l-histidine (PLH), and poly-l-arginine (PLA) formulations for light-directed small interference RNA (siRNA) gene silencing and messenger RNA (mRNA) delivery. We find that PLA formulations are suitable as siRNA and mRNA carriers in a strictly light-directed manner.
a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / p d p d t Summary Generation of singlet oxygen and estimation of its lifetime, the oxygen effect, the subcellular localization of photosensitizers and their photodegradation during photodynamic therapy (PDT), the effects of PDT on DNA and chromosomes were described in the first part of our review. In this review we discuss PDT in combination with other treatments (hyperthermia, ioniz-ing radiation, electrotherapy, chemotherapeutic drugs) or with other agents; cell interactions and bystander effects in PDT; the influence of PDT on the cytoskeleton; a novel technology, named photochemical internalisation (PCI), for light-induced delivery of macromolecules and 5-aminolevulinic acid (ALA) PDT. Lipophilic derivatives of ALA, instead of ALA itself, were proposed for induction of proto-porphyrin IX (PpIX) in the treatment of superficial lesions. Based on this research a Norwegian company PhotoCure ASA was founded in 1993 with the aim to commercialise photodynamic technologies developed at the Norwegian Radium Hospital, the largest comprehensive cancer centre in Northern Europe. The company has two products on the market: Metvix ® for treat-ment of basal cell carcinomas and actinic keratoses, and Hexvix ® for fluorescence diagnosis of bladder cancer. New ALA derivatives for fluorescence diagnosis and treatment of early-stage cancers in internal organs, e.g. colon cancer, are currently being investigated.
The use of polymers as synthetic non-viral carriers for introducing nucleic acids into cells
appears very appealing. Polymers can be generated in large quantities in chemically defined, non-antigenic
and non-immunogenic form. Aplethora of different chemical structures and polymer sizes may
be applied to tailor-made polymers with optimized characteristics for the extracellular delivery of
nucleic acid to the target tissue and the subsequent intracellular delivery into the target cells.
For the purpose of nucleic acid transfer, polymers have been applied for incorporating nucleic acids
into nanoparticles or microspheres. Alternatively, cationic polymers are applied as carriers for
complexing gene vectors into polyplexes. Polyplexes form spontaneously upon mixing negatively charged
nucleic acid with the polycationic polymer due to electrostatic interaction. This process can be
controlled to result in the formation of particles with defined virus-like sizes which efficiently
transfect cell cultures and also have shown encouraging gene transfer potential in in vivo administration.
With first-generation polymeric carriers, gene therapeutic effects have been demonstrated in animals,
although modest efficiencies and significant toxicity restrict broader therapeutic application. Key
issues for future optimization of polyplexes include improved specificity for the target tissue, enhanced
intracellular uptake, and reduced toxicity and immunogenicity. Novel cationic polymers have to be
made more biocompatible by reducing their potential for unspecific adverse interactions with the host,
and by designing them in abiodegradable form. “Smart” polymers and polymer conjugates
are being developed that in adynamic manner present virus-like delivery functions in the appropriate
phase of the gene delivery process.
Since the first generation of molecular machines including photoresponsive crown ethers and its analogues was reported by
Shinkai etal., a huge number of molecular machines exhibiting dynamic chemical and physical functions have been designed
and developed. On the other hand, non-viral vectors are desired to possess conflicting properties to associate with DNA until
reaching the nucleus as their final destination and dissociate from DNA there. In other words, non-viral vectors should work
as a sort of molecular machinery. To overcome this dilemma, recently, much attention is focused on the development of the
intelligent vectors, also called as ‘stimuli responsive vectors’ working as molecular machines. In this review, stimulus responsive
gene delivery systems in which some structural factors and/or physiological properties are regulated in response to extracellular
signals such as redox, pH, ultrasound, light, temperature, etc. are introduced as a new generation of non-viral vectors. These
extracellular signals such as ultrasound, light, and temperature can be potent stimuli capable of site-, timing-, and duration-specific
gene expression.
A major barrier within the field of non-viral gene therapy toward therapeutic strategies, e.g., tumor therapy, has been lack of appropriate specific delivery strategies to the intended target tissues or cells. In this chapter, we describe a protocol for light-directed delivery of nucleic acids through the use of photochemical internalization (PCI) technology. PCI is based on a photosensitizing compound that localizes to endocytic membranes. Upon illumination, the photosensitizing compound induces damage to the endocytic membranes, resulting in release of endocytosed material, i.e., nucleic acids into cytosol. The main benefit of the strategy described is the possibility for site-specific delivery of nucleic acids to a place of interest.
Photochemical internalization (PCI) is a technology based on a photosensitizer that photochemically destabilizes endosomal membranes after illumination, resulting in the release of endocytosed material into the cytosol. In this study, we investigated the potential of using polyethylenimine (PEI) for light-controlled delivery of small interfering RNA (siRNA) molecules via the endocytic pathway. PEI formulations with different molecular weights (MW) and chemical forms (linear [L]/branched [B]) were investigated for their capacity to deliver siRNA molecules with or without PCI at variable nitrogen/phosphorus (N/P) ratios and illumination doses. By targeting the S100A4 gene in an osteosarcoma cell model system, potent gene silencing was observed in samples treated with PCI compared with samples not treated with PCI. The effect of light-controlled gene silencing was dependent on several factors, including light-doses and MW, chemical form, as well as on the N/P ratio of the PEI formulations. This study demonstrates the first success in using PEI formulations as siRNA carriers for light-controlled gene silencing with the objective of future use in in vivo applications.
Photochemical internalization (PCI) is a novel technology for release of endocytosed macromolecules into the cytosol. The technology is based on the use of photosensitizers located in endocytic vesicles. Upon activation by light such photosensitizers induce a release of macromolecules from their compartmentalization in endocytic vesicles. PCI has been shown to increase the biological activity of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins, immunotoxins, plasmids, adenovirus, various oligonucleotides, dendrimer-based delivery of chemotherapeutica and unconjugated chemotherapeutica such as bleomycin and doxorubicin. This review will present the basis for the PCI concept and the most recent significant developments.
A successful cure of cancer by biopharmaceuticals with intracellular targets is dependent on both specific and sufficient delivery of the drug to the cytosol or nuclei of malignant cells. However, cytosolic delivery and efficacy of membrane-impermeable cancer therapeutics are often hampered by the sequestration and degradation of the drugs in the endolysosomal compartments. Hence, we developed photochemical internalization (PCI) as a site-specific drug delivery technology, which bursts the membrane of endocytic vesicles inducing release of entrapped drugs to the cytosol of light exposed cells. The principle of PCI has been demonstrated in >80 different cell lines and 10 different xenograft models of various cancers in different laboratories demonstrating its broad application potential. PCI-induced endosomal escape of protein- or nucleic acid-based therapeutics and some chemotherapeutics will be presented in this review. With a joint effort by life scientists the PCI technology is currently in a Phase I/II clinical trial with very promising initial results in the treatment of solid tumors.
The utilization of macromolecules in therapy of cancer and other diseases is becoming increasingly relevant. Recent advances in molecular biology and biotechnology have made it possible to improve targeting and design of cytotoxic agents, DNA complexes, and other macromolecules for clinical applications. To achieve the expected biological effect of these macromolecules, in many cases, internalization to the cell cytosol is crucial. At an intracellular level, the most fundamental obstruction for cytosolic release of the therapeutic molecule is the membrane-barrier of the endocytic vesicles. Photochemical internalization (PCI) is a novel technology for release of endocytosed macromolecules into the cytosol. The technology is based on the use of photosensitizers located in endocytic vesicles that upon activation by light induces a release of macromolecules from their compartmentalization in endocytic vesicles. PCI has been shown to potentiate the biological activity of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins (RIPs), gene-encoding plasmids, adenovirus, oligonucleotides, and the chemotherapeutic bleomycin. PCI has also been shown to enhance the treatment effect of targeted therapeutic macromolecules. The present protocol describes PCI of an epidermal growth factor receptor (EGFR)-targeted protein toxin (Cetuximab-saporin) linked via streptavidin-biotin for screening of targeted toxins as well as PCI of nonviral polyplex-based gene therapy. Although describing in detail PCI of targeted protein toxins and DNA polyplexes, the methodology presented in these protocols are also applicable for PCI of other gene therapy vectors (e.g., viral vectors), peptide nucleic acids (PNA), small interfering RNA (siRNA), polymers, nanoparticles, and some chemotherapeutic agents.
En se focalisant sur l'administration de médicaments dans et vers le système nerveux central et notamment pour le traitement du glioblastome, ce travail de thèse a eu pour but la mise en place d'outils expérimentaux et l'évaluation du comportement de nanovecteurs au cours du franchissement de barrières biologiques. Trois types de nanovecteurs de taille variant entre 20 et 100nm ont été appréhendés : des nanoparticules de polysaccharide, de PLGA et des nanocapsules lipidiques (LNC). Le comportement de ces objets vis-à-vis des éléments du sang a permis de définir que le revêtement par la transferrine de nanoparticules de PLGA et l'insertion de phospholipides ou de BSA dans des nanoparticules polysaccharidiques diminuait leur reconnaissance par le système réticulo-endothélial et améliorait leur temps de résidence plasmatique. Ces modifications de surface sont également associées à une possibilité d'internalisation dans les cellules cibles F98 de gliomes influencée essentiellement par la nature lipidique ou polymérique du vecteur. L'évaluation précise du comportement cellulaire et subcellulaire des LNC dans les cellules F98 a permis de démontrer que si la nature du vecteur est impliquée notamment en ce qui concerne le recrutement de voies d'endocytoses cholestéroldépendantes, la taille, corrélée au taux de surfactant véhiculé, est également impliquée. Les LNC de 20nm sont ainsi les plus aptes à permettre l'échappement lysosomal des principes actifs véhiculés et démontrent des activités pharmacologiques renforcées notamment pour ce qui concerne la mort cellulaire induite par le paclitaxel. Ces résultats établissent donc un lien original entre le comportement subcellulaire des vecteurs et la biodisponibilité des agents anticancéreux. De nouvelles potentialités de franchissement de barrières ligand- ou taille-dépendants ont été soulignées. Ces observations renforcent donc l'intérêt d'études comparatives permettant de rationaliser l'utilisation d'un vecteur donné pour un médicament et une cible donnés. Elles démontrent également tout l'intérêt d'établir des justifications entre le comportement biologique et la pertinence thérapeutique des nanovecteurs.
To present a new method called magnetolipofection which can transfect cells in a specific area of the retinal pigment epithelium (RPE) by magnetic force as a non-viral gene transfection.
ARPE-19 (a human RPE cell line) cells were cultured with a mixture of cationic lipid, plasmid DNAs and magnetite nanoparticles. A sheet of ARPE-19 cells was transfected in the vertical direction by placing a magnet under the centre of the culture plate. Horizontal gene transfection was also performed.
When magnetolipofection was performed in the vertical direction, there was a significantly larger number of green fluorescent protein (GFP)-positive cells where the magnet was placed than in the peripheral area, and the number was equivalent to the number transfected with Lipofectamine2000. In the horizontal direction, there was also a significantly larger number of GFP-positive cells, but there was almost no gene transfer detected using Lipofectamine2000.
The area of gene transfection can be controlled by the placement of a magnet in the area selected to be transfected in vitro by magnetolipofection. This method can be used to transfect RPE cells in selected areas which should be helpful for experimental and clinical applications.
After intravenous injection, DNA complexed with asialoglycoprotein-polylysine conjugates is endocytosed by hepatocytes via asialoglycoprotein receptors and is expressed transiently. Long term persistence and expression occurs when partial hepatectomy is performed after gene delivery. To determine the intracellular location of the persisting DNA, we transferred a plasmid expressing bacterial chloramphenicol acetyltransferase into the liver of rats in vivo by asialoglycoprotein receptor-mediated endocytosis. The internalized DNA was measured by Southern blot. Twenty min after administration, 80-85% of the plasmid appeared in the liver, 80% of which was within hepatocytes (12,000-18,000 copies/hepatocyte). In sham-operated control rats, the transgene concentration decreased to 8-12 and 2-4% of the initial levels in 4 and 24 h, respectively, and became undetectable at 7 days. In rats subjected to 66% hepatectomy 20 min after DNA administration, 20, 9, and 7% of the plasmid in the residual liver persisted at 4 h, 24 h, and 7 days, respectively. Liver homogenates were fractionated by differential centrifugation and Percoll gradient centrifugation. In 66% hepatectomized rats, the plasmid persisted in an undegraded, transfection-competent form in plasma membrane/endosome-enriched fractions throughout the duration of the experiment (7 days), indicating that cytoplasmic vesicles are the main site of persistence of the endocytosed DNA.
The process by which viruses destabilize endosomal membranes in an acidification dependent manner has been mimicked with synthetic peptides that are able to disrupt liposomes, erythrocytes, or endosomes of cultured cells. Peptides containing the 20 amino-terminal amino acid sequence of influenza virus hemagglutinin as well as acidic derivatives showed erythrocyte lysis activity only when peptides were elongated by an amphipathic helix or by carboxyl-terminal dimerization. Interestingly, peptides consisting of the 23 amino-terminal amino acids of influenza virus hemagglutinin were also active in erythrocyte lysis. When peptides were in corporated into DNA complexes that utilize a receptor-mediated endocytosis pathway for uptake into cultured cells, either by ionic interaction with positively charged polylysine-DNA complexes or by a streptavidin-biotin bridge, a strong correlation between pH-specific erythrocyte disruption activity and gene transfer was observed. A high-level expression of luciferase or interleukin-2 was obtained with optimized gene transfer complexes in human melanoma cells and several cell lines.
One limit to successful receptor-mediated gene delivery is the exit of the endocytosed material from the endosome. We demonstrate here the delivery of marker genes to tissue culture cells using a modification of the receptor-mediated gene delivery technique that exploits the endosomolytic activity of defective adenovirus particles. In particular, greater than 90% of the transfected-cell population is found to express a beta-galactosidase gene, and, most importantly, this high level of expression can be obtained with psoralen-inactivated virus particles. Furthermore, because the delivered gene is not carried within the genome of the adenovirus particle, the size constraints are relieved, and we can, therefore, show the delivery of a 48-kilobase cosmid DNA molecule.
Plasmid DNA/glycosylated polylysine complexes were used to transfer in vitro a luciferase reporter gene into human hepatoma cells by a receptor-mediated endocytosis process. HepG2 cells which express
a galactose specific membrane lectin were efficiently and selectively transfected with pSV2Luc/lactosylated polylysine complexes
in a sugar dependent manner: I) HepG2 cells which do not express membrane lectin specific for mannose were quite poorly transfected
with pSV2Luc/mannosylated polylysine complexes, II) HeLa cells which do not express membrane lectin specific for galactose
were not transfected with pSV2Luc/lactosylated polylysine complexes. The transfection efficiency of HepG2 cells with pSV2Luc/lactosylated
polylysine complexes was greatly enhanced either in the presence of chloroquine or in the presence of a fusogenic peptide.
A 22-residue peptide derived from the influenza virus hemagglutinin HA2 N-terminal polypeptide that mimics the fusogenic activity
of the virus, was selected. In the presence of the fusogenic peptide, the luciferase activity in HepG2 cells was 10 fold larger
than that of cells transfected with pSV2Luc/lactosylated polylysine complexes in the presence of chloroquine.
We are developing efficient methods for gene transfer into tissue culture cells. We have previously shown that coupling of a chimeric adenovirus with polylysine allowed the construction of an adenovirus-polylysine-reporter-gene complex that transferred the transporter gene with great efficiency into HeLa cells. We have now explored simpler, biochemical means for coupling adenovirus to DNA/polylysine complexes and show that such complexes yield virtually 100% transfection in tissue culture cell lines. In these methods adenovirus is coupled to polylysine, either enzymatically through the action of transglutaminase or biochemically by biotinylating adenovirus and streptavidinylating the polylysine moiety. Combination complexes containing DNA, adenovirus-polylysine, and transferrin-polylysine have the capacity to transfer the reporter gene into adenovirus-receptor- and/or transferrin-receptor-rich cells.
Gene transfer may be accomplished by the receptor-mediated endocytosis pathway using transferrin-polylysine conjugates. For some target cells, however, gene transfer by this vector is extremely limited, despite the presence of the appropriate surface receptors, a phenomenon attributed to lysosomal degradation of endosome-internalized conjugate-DNA complexes. To enhance DNA escape from the cell vesicle system and thus augment gene transfer by this route, we have used the capacity of adenoviruses to disrupt endosomes as part of their entry mechanism. Adenoviral infection augmented levels of gene transfer by transferrin-polylysine conjugates in a dose-dependent manner: levels of gene transfer of greater than 2000-fold above baseline were achieved. Use of the adenovirus in this context allowed enhanced levels of gene transfer in a variety of target cells, including cell lines otherwise refractory to gene transfer by transferrin-polylysine conjugates. This augmentation was based on adenoviral-mediated vesicle disruption, a process independent of viral gene expression. Thus, the development of specific mechanisms to effect release from the endosome in combination with gene transfer by the receptor-mediated endocytosis pathway will increase the utility of this delivery system by allowing high levels of gene expression in target cells.
Difficulties with the nondestructive delivery of macromolecules into living cells have limited the potential applications of antibodies, genes, enzymes, peptides, and antisense oligonucleotides in biology and medicine. We have found, however, that the natural endocytosis pathway for the vitamin folate can be exploited to nondestructively introduce macromolecules into cultured cells if the macromolecule is first covalently linked to folate. Thus, treatment of KB cells with folate-conjugated ribonuclease, horseradish peroxidase, serum albumin, IgG, or ferritin allowed delivery of greater than 10(6) copies of the macromolecules within a 2-hr period. Cytochemical staining using 4-chloro-1-naphthol further demonstrated that the horseradish peroxidase retained activity for at least 6 hr after internalization. Since folate is an essential vitamin required in substantial quantities by virtually all cells, these observations may open the possibility of scientific and medical applications for many of the above macromolecules.
Most current gene transfer methods function satisfactorily in specialized systems involving established cell lines but are often not applicable with nonadherent, primary hematopoietic cells, which are notoriously difficult to transfect. To approach this problem, we have investigated an alternative method of gene transfer, "transferrinfection," in which DNA complexed to transferrin-polycation conjugates is introduced into cells by receptor-mediated endocytosis [Wagner, E., Zenke, M., Cotten, M., Beug, H. & Birnstiel, M. L. (1990) Proc. Natl. Acad. Sci. USA 87, 3410-3414]. We show here that transferrin-polylysine and transferrin-protamine, when complexed to plasmid DNA containing a luciferase reporter gene, is efficiently bound and moved into avian erythroblasts by endocytosis. Successful transfer and expression of the luciferase reporter gene depends on specific interaction of the transferrin-polylysine-DNA complex with the transferrin receptor and occurs in a significant fraction (greater than 95%) of the cells. Gene transfer efficiency by transferrinfection is lower than with an optimized DEAE-dextran transfection method but reaches similar efficiencies when the cells are treated with chloroquine. Because the procedure in the absence of chloroquine is completely nontoxic to cells, a constant expression level of transferred genes may be maintained by repeated additions of transferrin-polylysine-DNA complex. In addition, the usefulness of transferrinfection for gene transfer into primary hematopoietic cells is demonstrated.
We have subverted a receptor-mediated endocytosis event to transport genes into human leukemic cells. By coupling the natural iron-delivery protein transferrin to the DNA-binding polycations polylysine or protamine, we have created protein conjugates that bind nucleic acids and carry them into the cell during the normal transferrin cycle [Wagner, E., Zenke, M., Cotten, M., Beug, H. & Birnstiel, M. L. (1990) Proc. Natl. Acad. Sci. USA 87, 3410-3414]. We demonstrate here that this procedure is useful for a human leukemic cell line. We enhanced the rate of gene delivery by (i) increasing the transferrin receptor density through treatment of the cells with the cell-permeable iron chelator desferrioxamine, (ii) interfering with the synthesis of heme with succinyl acetone treatment, or (iii) stimulating the degradation of heme with cobalt chloride treatment. Consistent with gene delivery as an endocytosis event, we show that the subsequent expression in K-562 cells of a gene included in the transported DNA depends upon the cellular presence of the lysosomotropic agent chloroquine. By contrast, monensin blocks "transferrinfection," as does incubation of the cells at 18 degrees C.
In this paper we describe stably transfected rat cell lines which harbour either the human cytomegalovirus (HCMV) immediate early (IE) gene encoding the 72K IE nuclear antigen (IEA) or the bacterial chloramphenicol acetyltransferase (CAT) gene both under transcriptional control of the HCMV IE enhancer-promoter (-484 to -19 relative to the IE cap site, +1). In these cell lines IE gene or CAT gene expression is repressed but can be induced by heat-shock, by sodium arsenite and by inhibitors of protein synthesis such as cycloheximide (CH). In addition, we present evidence suggesting that CH-mediated activation is cell cycle-dependent. Thus CH-mediated induction of the 72K IEA as well as CAT gene expression was impaired and accumulation of mRNAs did not occur when cellular DNA synthesis was inhibited. Activation of IE genes by CH occurred almost exclusively in those cells which were in S-phase. In contrast, activation of gene expression by sodium arsenite occurred independently of cellular DNA synthesis and was not restricted to cells in S-phase. The data are consistent with, but not proof of, the hypothesis that the activation of IE transcription, brought about by inhibition of protein synthesis, resulted from a disturbed chromatin conformation due to DNA synthesis continuing in the absence of a supply of chromatin-organizing proteins. The possible relevance of these observations with regard to HCMV latency and reactivation is discussed.
We have used a particular folate receptor, which is overexpressed in tumor cells, for targeted DNA delivery into these cell types. This folate receptor internalizes folate through caveolae by a process named potocytosis, which is distinct from endocytosis, through clathrin-coated pits. When folate conjugated to poly-L-lysine was used to deliver the E. coli beta-galactosidase gene into tumor cells overexpressing the folate receptor, only low levels of beta-galactosidase activity were detectable. When a replication-defective adenovirus was coincubated with the DNA/folate complexes, 20 to 30% of the cells stained blue with X-gal and a 1000-fold increase of beta-galactosidase activity was observed. Thus, for high efficient DNA delivery and gene expression via the caveolae system, a potosomal disruption agent is needed. Furthermore, folate-mediated DNA delivery is restricted to tumor cells that highly overexpress the folate receptor, which will permit future development of tumor cell-specific delivery of toxic genes for cancer gene therapy.
Cationic lipids are widely used for gene transfer in vitro and show promise as a vector for in vivo gene therapy applications. However, there is limited understanding of the cellular and molecular mechanisms involved. We investigated the individual steps in cationic lipid-mediated gene transfer to cultured cell lines. We used DMRIE/DOPE (a 1:1 mixture of N-[1-(2,3-dimyristyloxy) propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide (DMRIE) and dioleoyl phosphatidylethanolamine (DOPE) as a model lipid because of its efficacy and because it is being used for clinical trials in humans. The data show that cationic lipid-mediated gene transfer is an inefficient process. Part of the inefficiency may result from the fact that the population of lipid-DNA complexes was very heterogeneous, even under conditions that have been optimized to produce the best transfection. Inefficiency was not due to inability of the complex to enter the cells because most cells took up the DNA. However, in contrast to previous speculation, the results indicate that endocytosis was the major mechanism of entry. After endocytosis, the lipid-DNA aggregated into large perinuclear complexes, which often showed a highly ordered tubular structure. Although much of the DNA remained aggregated in a vesicular compartment, there was at least a small amount of DNA in the cytoplasm of most cells. That observation plus results from direct injection of DNA and lipid-DNA into the nucleus and cytoplasm indicate that movement of DNA from the cytoplasm to the nucleus may be one of the most important limitations to successful gene transfer. Finally, before transcription can occur, the data show that lipid and DNA must dissociate. These results provide new insights into the physical limitations to cationic lipid-mediated gene transfer and suggest that attention to specific steps in the cellular process may further improve the efficiency of transfection and increase its use in a number of applications.
The process by which viruses destabilize endosomal membranes in an acidification-dependent manner has been mimicked with synthetic peptides that are able to disrupt liposomes, erythrocytes, or endosomes of cultured cells. Peptides containing the 20 amino-terminal amino acid sequence of influenza virus hemagglutinin as well as acidic derivatives showed erythrocyte lysis activity only when peptides were elongated by an amphipathic helix or by carboxyl-terminal dimerization. Interestingly, peptides consisting of the 23 amino-terminal amino acids of influenza virus hemagglutinin were also active in erythrocyte lysis. When peptides were incorporated into DNA complexes that utilize a receptor-mediated endocytosis pathway for uptake into cultured cells, either by ionic interaction with positively charged polylysine-DNA complexes or by a streptavidin-biotin bridge, a strong correlation between pH-specific erythrocyte disruption activity and gene transfer was observed. A high-level expression of luciferase or interleukin-2 was obtained with optimized gene transfer complexes in human melanoma cells and several cell lines.
The application of cationic liposome reagents has advanced DNA and mRNA transfection research in vitro, and data are accumulating which show their utility for in vivo gene transfer. However, chemical structure-activity data leading to a better mechanistic understanding of their biological activity is still limited. Most of the cationic lipid reagents in use today for this application are formulated as liposomes containing two lipid species, a cationic amphiphile and a neutral phospholipid, typically dioleoylphosphatidylethanolamine (DOPE). The studies reported here examine the effects of some systematic chemical structural changes in both of these lipid components. Cationic and neutral phospholipids were formulated together as large multilamellar vesicles (MLV) or small sonicated unilamellar vesicles (SUV) in water, and each formulation was assayed quantitatively in 96-well microtiter plates under 64 different assay conditions using COS.7 cells and an RSV-beta-galactosidase expression plasmid. The cationic lipid molecules used for these studies were derived from a novel series of 2,3-dialkyloxypropyl quaternary ammonium compounds containing a hydroxyalkyl moiety on the quaternary amine. A homologous series of dioleylalkyl (C18:1) compounds containing increasing hydroxyalkyl chain lengths on the quaternary amine were synthesized, formulated with 50 mol % DOPE, and assayed for transfection activity. The order of efficacy was ethyl > propyl > butyl > pentyl > 2,3-dioleyloxypropyl-1-trimethyl ammonium bromide (DOTMA). DOTMA, which is commercially available under the trademark Lipofectin Reagent, lacks a hydroxyalkyl moiety on the quaternary amine. A homologous series of hydroxyethyl quaternary ammonium derivatives with different alkyl chain substitutions were synthesized, formulated with 50 mol % DOPE, and assayed in the transfection assay. The order of transfection efficacy was dimyristyl (di-C14:0) > dioleyl (di-C18:1) > dipalmityl (di-C16:0) > disteryl (di-C18:0). The addition of 100 microM chloroquine in the transfection experiment enhanced the activity of the dioleyl compound by 4-fold and decreased the activity of the dimyristyl compound by 70%. For each of the compounds and formulations examined in this report, large multilamellar vesicles (MLV; diameter 300-700 nm) were more active than small unilamellar vesicles (SUV; diameter 50-100 nm). The neutral phospholipid requirements for transfection activity in COS.7 cells with these cationic lipid molecules were examined.(ABSTRACT TRUNCATED AT 400 WORDS)
We have combined a receptor-mediated DNA delivery system with the endosomal lysis ability of adenovirus and shown that DNA can be delivered into primary hepatocytes, resulting in a high level of gene expression. When asialoorosomucoid conjugated with poly(L-lysine) was used to deliver the Escherichia coli beta-galactosidase gene into primary hepatocytes through binding with the hepatic asialoglycoprotein receptor, only a low level of beta-galactosidase was detectable, with less than 0.1% of the hepatocytes being transfected. This level of activity can be greatly enhanced by the cointernalization of the DNA.protein complex with a replication-defective adenovirus, resulting in 100% of the hepatocytes staining blue with 5-bromo-4-chloro-3-indolyl beta-D-galactoside. Quantitative analysis of beta-galactosidase expression also showed a 1000-fold enhancement of activity. To test the applicability of this DNA delivery system for the correction of phenylketonuria, a metabolic disorder that causes severe mental retardation in children, we have delivered the human phenylalanine hydroxylase (PAH) gene to hepatocytes derived from a PAH-deficient mouse strain and demonstrated complete reconstitution of enzymatic activity. This method shows great promise for efficient gene delivery to the liver for correction of hepatic disorders.
Molecular conjugate vectors may be constructed that accomplish high efficiency gene transfer by the receptor-mediated endocytosis pathway. In order to mediate escape from lysosomal degradation, we have incorporated adenoviruses into the functional design of the conjugate. In doing so, however, we have introduced an additional ligand, which can bind to receptors on the cell surface, undermining the potential for cell specific targeting. To overcome this, we have treated the adenovirus with a monoclonal anti-fiber antibody, which renders the virus incapable of binding to its receptor. The result is a multi-functional molecular conjugate vector, which has preserved its binding specificity while at the same time being capable of preventing lysosomal degradation of endosome-internalized conjugate-DNA complexes. This finding indicates that adenoviral binding is not a prerequisite for adenoviral-mediated endosome disruption.
After intravenous injection, DNA complexed with asialoglycoprotein-polylysine conjugates is endocytosed by hepatocytes via asialoglycoprotein receptors and is expressed transiently. Long term persistence and expression occurs when partial hepatectomy is performed after gene delivery. To determine the intracellular location of the persisting DNA, we transferred a plasmid expressing bacterial chloramphenicol acetyltransferase into the liver of rats in vivo by asialoglycoprotein receptor-mediated endocytosis. The internalized DNA was measured by Southern blot. Twenty min after administration, 80-85% of the plasmid appeared in the liver, 80% of which was within hepatocytes (12,000-18,000 copies/hepatocyte). In sham-operated control rats, the transgene concentration decreased to 8-12 and 2-4% of the initial levels in 4 and 24 h, respectively, and became undetectable at 7 days. In rats subjected to 66% hepatectomy 20 min after DNA administration, 20, 9, and 7% of the plasmid in the residual liver persisted at 4 h, 24 h, and 7 days, respectively. Liver homogenates were fractionated by differential centrifugation and Percoll gradient centrifugation. In 66% hepatectomized rats, the plasmid persisted in an undegraded, transfection-competent form in plasma membrane/endosome-enriched fractions throughout the duration of the experiment (7 days), indicating that cytoplasmic vesicles are the main site of persistence of the endocytosed DNA.
We have developed a lipidic gene transfer vector, LPDII, where DNA was first complexed to polylysine at a ratio of 1:0.75 (w/w) and then entrapped into folate-targeted pH-sensitive anionic liposomes composed of dioleoyl phosphatidylethanolamine (DOPE)/cholesteryl hemisuccinate/folate-polyethlene glycol-DOPE (6:4:0.01 mol/mol) via charge interaction. LPDII transfection of KB cells, a cell line overexpressing the tumor marker folate receptor, was affected by both the lipid to DNA ratio and the lipid composition. At low lipid to DNA ratios (e.g. 4 and 6), LPDII particles were positively charged; transfection and cellular uptake levels were independent of the folate receptor and did not require a pH-sensitive lipid composition. Meanwhile, transfection and uptake of negatively charged LPDII particles, i.e. those with high lipid to DNA ratios (e.g. 10 and 12), were folate receptor-dependent and required a pH-sensitive lipid composition. The transfection activity of LPDII was lost when the inverted cone-shaped DOPE was replaced by dioleoyl phosphatidylcholine. LPDII particles with lipid to DNA ratios of 4, 6, 10, and 12 were approximately 20-30 times more active than DNA.3-beta-[N-(N',N'-dimethylethane)carbamoyl]cholesterol cationic liposome complexes in KB cells and were much less cytotoxic. On the sucrose gradient, LPDII particles had a migration rate in between those of the free DNA and the DNA.polylysine complex. An electron micrograph of LPDII showed a structure of spherical particles with a positively stained core enclosed in a lipidic envelope with a mean diameter of 74 +/- 14 nm. This novel gene transfer vector may potentially be useful in gene therapy for tumor-specific delivery.
Particle-mediated (gene gun) in vivo delivery of the murine interleukin 12 (IL-12) gene in an expression plasmid was evaluated for antitumor activity. Transfer of IL-12 cDNA into epidermal cells overlying an implanted intradermal tumor resulted in detectable levels (266.0 +/- 27.8 pg) of the transgenic protein at the skin tissue treatment site. Despite these low levels of transgenic IL-12, complete regression of established tumors (0.4-0.8 cm in diameter) was achieved in mice bearing Renca, MethA, SA-1, or L5178Y syngeneic tumors. Only one to four treatments with IL-12 cDNA-coated particles, starting on day 7 after tumor cell implantation, were required to achieve complete tumor regression. This antitumor effect was CD8+ T cell-dependent and led to the generation of tumor-specific immunological memory. By using a metastatic P815 tumor model, we further showed that a delivery of IL-12 cDNA into the skin overlying an advanced intradermal tumor, followed by tumor excision and three additional IL-12 gene transfections, could significantly inhibit systemic metastases, resulting in extended survival of test mice. These results suggest that gene gun-mediated in vivo delivery of IL-12 cDNA should be further developed for potential clinical testing as an approach for human cancer gene therapy.
Primary human fibroblasts and a series of cell lines (A549, BNL CL.2, H225, NIH 3T3 and Rat-1) are efficiently transfected by using positively charged complexes of plasmid DNA and transferrin-polylysine or polylysine in the presence of glycerol (1 molar to 1.8 molar, depending on the cell type). An increase in gene expression of up to several-hundredfold (compared to complexes without glycerol) is obtained if the transfection mixture is incubated with the cells for 3-4 h at 37 degrees C. This simple method has been used for transient expression of luciferase, beta-galactosidase and interleukin-2, and also for the generation of stably transfected cells.
The presence of about 1.2 M glycerol during transfection with DNA/transferrin-polylysine and DNA/polylysine complexes dramatically increases transgene expression in a variety of cell types, provided that the complexes have an excess of polylysine. We have characterized this phenomenon using a human melanoma cell line (H225). The addition of 1.2 M glycerol to the transfection medium has no influence on the internalization of DNA complexes or on the promoter activity used to direct reporter gene expression. Neither prenor postincubation of the cells with glycerol results in a notable increase in transgene expression. Bafilomycin A1 and chloroquine, two drugs affecting the endosomal pathway, both influenced transgene expression, indicating that glycerol acts on internal vesicles. Glycerol and polylysine synergized in their ability to lyse erythrocytes as well as internal vesicles (microsomes) isolated from H225 cells, indicating that the glycerol effect is due to a labilization of vesicular membranes, which facilitates membrane disruption by polylysine. Our current model suggests that the excess of polylysine in the DNA complexes disrupts vesicular membranes in the presence of glycerol, thus allowing the release of DNA complexes into the cytoplasm.
To develop a nonviral gene delivery system for treatment of diseases, our strategy is to construct DNA complexes with short synthetic peptides that mimic the functions of viral proteins. We have designed and synthesized two peptides which emulate viral functions - a DNA condensing agent, YKAK(8)WK, and an amphipathic, pH-dependent endosomal releasing agent, GLFEALLELLESLWELLLEA. The active gene delivery complex was constructed step-wise through a spontaneous self-assembly process involving oppositely charged, electrostatic interactions. To assemble DNA-peptide complexes with different overall net charges, only the negative charges of DNA phosphate, the positive charges of the 10 epsilon-amino groups of YKAK(8)WK and the negative charges of the 5 gamma-carboxyl groups of GLFEALLELLESLWELLLEA were considered. In the first step, negatively charged DNA was rapidly-mixed with an excess of YKAK(8)WK to form positively charged DNA-YKAK(8)WK complexes, which gave little gene transfer. In the second step and to form the active complex,the cationic DNA complex was rapidly mixed with spontaneously incorporated through electrostatic interactions. Transfection using these complexes of CMV-luc, YKAK(8)WK and GLFEALLELLESLWELLLEA gave high-levels of gene expression in a variety of cell lines. These simple DNA complexes, which contain only three molecularly defined components, have general utility for gene delivery and can replace viral vectors and cationic lipids for some applications in gene therapy.
Cationic liposomes provide a means to introduce genes into cells both ex vivo and in vivo. In the past few years their use has been described in several tissues, e.g. lungs, liver, endothelium, brain. In this study we evaluated a commercially available poly-cationic liposome formulation in delivering a reporter gene into cultured myogenic cell lines from mouse and rat, and primary fetal human myoblasts. We also examined the effect of serum on liposome-mediated transfection and designed a new procedure to enhance transfection efficiency, based on the pre-condensation of plasmid DNA with polylysine. Polylysine pre-condensation was particularly effective when transfecting the cells in the presence of serum, a finding that could be significant for in vivo transfections.
We have synthesized a linear, bifunctional peptide that comprises an integrin-targeting domain containing an arginine-glycine-aspartic acid tripeptide motif and a DNA-binding moiety consisting of a short stretch of 16 lysine residues. This peptide can form distinctive, condensed complexes with DNA and is capable of mediating its delivery and expression in a variety of mammalian cells in culture. Internalization is mediated by cell surface integrin receptors via a mechanism that is known to be phagocytic. We have analyzed the relationship between DNA and peptide and have investigated the conditions suitable for optimal gene delivery. The formation of condensed peptide DNA complexes leads to resistance to nuclease degradation. The level of reporter gene expression obtained is dependent on the peptide-to-DNA ratio and is enhanced in the presence of the endosomal buffer chloroquine, polyethyleneimine, and deactivated adenovirus during gene delivery. Under optimal conditions the levels of reporter gene expression obtained approach or even exceed those obtained with DNA delivered with the commercial liposome Lipofectamine. The ability to produce an efficient gene delivery system using small, easily modified, and well-defined constructs that have no constraint of particle size demonstrates the advantages of integrin-targeting peptides for gene transfer.
A pH-responsive peptide fragment modelled on the influenza virus haemagglutinin (INF7-SGSC) can promote the transfectional activity of poly(L)-lysine (pLL)/DNA complexes against 293 cells. Chloroquine also promotes transfection, but the combination of INF7-SGSC and chloroquine gives an increased, synergistic, transfectional activity. This was unexpected since the supposed modes of action of these two agents are expected to be incompatible. Microinjection of pLL/DNA complexes into the cytoplasm of Xenopus oocytes produced greater gene expression than microinjection of free DNA, possibly reflecting nuclear-homing or protection from degradation by cytoplasmic nucleases. However, pretreatment of complexes with INF7-SGSC (but not chloroquine) before cytoplasmic microinjection promoted gene expression still further. When pLL/DNA complexes were injected directly into the nucleus, INF7-SGSC again increased gene expression. The mechanism of post-endosomal action of INF7-SGSC is unknown, but could reflect its polyanionic nature, possibly enhancing intranuclear dissociation of the complexes. Whatever the mechanism, it appears that INF7-SGSC mediates two effects-one probably endosomal and the second post-endosomal, the latter showing a synergistic transfection interaction with chloroquine.
The therapeutic usefulness of macromolecules, such as in gene therapy, is often limited by an inefficient transfer of the macromolecule to the cytosol and a lack of tissue-specific targeting. The possibility of photochemically releasing macromolecules from endosomes and lysosomes into the cytosol was examined. Endocytosed macromolecules and photosensitizer were exposed to light and intracellular localization and the expression of macomolecules in the cytosol was analyzed. This novel technology, named photochemical internalization (PCI), was found to efficiently deliver type I ribosome-inactivating proteins, horseradish peroxidase, a p21ras-derived peptide, and a plasmid encoding green fluorescent protein into cytosol in a light-dependent manner. The results presented here show that PCI can induce efficient light-directed delivery of macromolecules into the cytosol, indicating that PCI may have a variety of useful applications for site-specific drug delivery, e.g., in gene therapy, vaccination, and cancer treatment.
Among the nonviral techniques for gene transfer in vivo, the direct injection of plasmid DNA into muscle is simple, inexpensive, and safe. Applications of this method have been limited by the relatively low expression levels of the transferred gene. We investigated the applicability of in vivo electroporation for gene transfer into muscle, using plasmid DNA expressing interleukin-5 (IL-5) as the vector. The tibialis anterior muscles of mice were injected with the plasmid DNA, and then a pair of electrode needles were inserted into the DNA injection site to deliver electric pulses. Five days later, the serum IL-5 levels were assayed. Mice that did not receive electroporation had serum levels of 0.2 ng/ml. Electroporation enhanced the levels to over 20 ng/ml. Histochemical analysis of muscles injected with a lacZ expression plasmid showed that in vivo electroporation increased both the number of muscle fibers taking up plasmid DNA and the copy number of plasmids introduced into the cells. These results demonstrate that gene transfer into muscle by electroporation in vivo is more efficient than simple intramuscular DNA injection.
In recent years, there have been a number of technological breakthroughs
that have allowed for clinical trials in gene therapy to be initiated.
In combination with the genome initiative, the potential for new
therapeutics is limitless. Although an enormous amount of information
has been obtained in a relatively short period of time, gene therapy is
not yet ready for wide-scale practice. Some of the successes and
obstacles that remain are summarized in this report.
A close interaction between chemistry and medicine is offered by cationic liposome research. Cationic liposomes are able to deliver nucleic acids to cells and are projected to be core technology for the emerging discipline of gene therapy. The problems that still have to be faced are presented here along with potential solutions.
Receptor-mediated gene transfer is a promising gene delivery technique. It employs a DNA-binding polycation, such as polylysine, to compact plasmid DNA to a size that can be taken up by cells (<100-200 nm). To allow internalization by receptor-mediated endocytosis, cell binding ligands, such as asialoglycoproteins or galactose for hepatocytes, anti-CD3 and anti-CD5 for T-cells, and transferrin, have been covalently attached to polylysine. Intracellular barriers for successful gene transfer include release of DNA complexes from endosomes or lysosomes, nuclear import of DNA complexes, and disassembly of the DNA-polylysine particles. Release of particles from internal vesicles has been achieved by the addition of lysosomotropic agents or glycerol to the transfection medium, or by the incorporation of endosomolytic compounds, such as viruses or membrane active peptides. This technique has already been used to transfect certain organs in vivo, including liver and lung
Transferrin, an iron-transporting serum glycoprotein, is efficiently taken up into cells by the process of receptor-mediated endocytosis. Transferrin receptors are found on the surface of most proliferating cells, in elevated numbers on erythroblasts and on many kinds of tumors. The efficient cellular mechanism for uptake of transferrin has been subverted for the delivery of low-molecular-weight drugs, protein toxins, and liposomes by linkage of these agents to transferrin or to anti-transferrin receptor antibodies. Linkage may be via chemical conjugation procedures or by the generation of chimeric fusion proteins. Transferrin conjugated to DNA-binding compounds (e.g. polycations or intercalating agents) has been successfully used for the import of DNA molecules into cells. High-level gene expression is obtained only if endosome-disruptive agents such as influenza hemagglutinin peptides or adenovirus particles are included which release the DNA complex from intracellular vesicles into the cytoplasm.
Hox genes, which play key roles in the development of body plans, have
been described from a variety of metazoans. Here we report the presence
of Hox class genes that are typical of triploblasts in Myxozoa, formerly
a protozoan taxon. This finding confirms Myxozoa's phylogenetic affinity
with the Bilateria and reveals an extreme example of parasitic
degeneracy.
Activation of transcription of the Egr-1 gene by X-rays is regulated by the promoter region of this gene. We linked the radiation-inducible promoter region of the Egr-1 gene to the gene encoding the radiosensitizing and tumoricidal cytokine, tumour necrosis factor-alpha (TNF-alpha) and used a replication-deficient adenovirus to deliver the Egr-TNF construct to human tumours growing in nude mice. Combined treatment with Ad5.Egr-TNF and 5,000 cGy (rad) resulted in increased intratumoral TNF-alpha production and increased tumour control compared with treatment with Ad5.Egr-TNF alone or with radiation alone. The increase in tumour control was achieved without an increase in normal tissue damage when compared to tissue injury from radiation alone. Control of gene transcription by ionizing radiation in vivo represents a novel method of spatial and temporal regulation of gene-based medical treatments.
To assess the potential value of the 6-day subrenal capsule (SRC) assay in preclinical evaluation of new drugs using serially xenografted human tumors as source of tumor tissue, we studied the response of 31 human tumor lines (8 malignant melanomas, 12 sarcomas, 9 lung carcinomas and 2 colon carcinomas) to relevant standard drugs and to a new imidazotetrazine, Mitozolomide. Mitozolomide was found to be the most active drug tested in 50% of the lung carcinomas and as active as CCNU in melanomas. The activity of the standard anticancer drugs against subrenal grafts closely resembled the patterns seen with the same tumors in the clinic. In further attempts to validate the procedure, sensitivity profiles of some tumors were concurrently determined in the subcutaneous (s.c.) nude mouse model. In 11 out of the 12 tumors, the two assays selected the same drug as being the most active and in most of these tumors the two procedures gave the same ranking for the different drugs. Also, when the relative sensitivities of a series of melanoma xenografts to each of two drugs (DTIC and CCNU) were tested, the two assays gave the same ranking of the xenografts for each drug. The concordance between the two assays and the fact that the s.c. nude mouse assay reflects the chemosensitivity of the parent tumor in patients, suggest that the application of the 6-day SRC assay to xenografted tumors is a valid and useful procedure permitting rapid preclinical evaluation of new drugs to be carried out at relatively low cost.
— Spectral transmittance of 400–865 nm radiation through various human structures, including the skull with scalp, the chest wall, abdominal wall and scrotum, is presented. There is essentially no visible light of wavelengths shorter than 500 nm transmitted through the chest or the abdominal wall. In contrast, 10−5–10−4 of blue light can reach the brain and testes. Transmittance of all tissues increases progressively with wavelength from 600 to 814nm. The maximal transmittances are 10−2–10−1 for skulls and scrota, and 10−3–10−2 for chest and abdominal walls. Tissue thickness, optical absorption and scattering are major influencing factors.
A tetrazolium salt has been used to develop a quantitative colorimetric assay for mammalian cell survival and proliferation. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision. No washing steps are used in the assay. The main advantages of the colorimetric assay are its rapidity and precision, and the lack of any radioisotope. We have used the assay to measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis.
Tumor cells expressing the herpes simplex virus thymidine kinase (HSV-TK) gene are sensitive to the drug ganciclovir (GCV). We demonstrate here that HSV-TK-positive cells exposed to GCV were lethal to HSV-TK-negative cells as a result of a "bystander effect." HSV-TK-negative cells were killed in vitro when the population of cultured cells contained only 10% HSV-TK-positive cells. The mechanism of this "bystander effect" on HSV-TK-negative cells appeared to be related to the process of apoptotic cell death when HSV-TK-positive cells were exposed to GCV. Flow cytometric and electron microscopic analyses suggested that apoptotic vesicles generated from the dying gene-modified cells were phagocytized by nearby, unmodified tumor cells. Prevention of apoptotic vesicle transfer prevented the bystander effect. The toxic effect of HSV-TK-positive cells on HSV-TK-negative cells was reproduced in an in vivo model. A mixed population of tumor cells consisting of HSV-TK-positive and HSV-TK-negative cells was inoculated s.c. into mice. Regression of the tumor mass occurred when the inoculum consisted of as few as 10% HSV-TK-expressing tumor cells. The bystander effect was also demonstrated in i.p. tumor studies. Initial experiments demonstrated that prolonged survival (> 70 days) occurred when a mixture containing 50% HSV-TK-positive and 50% HSV-TK-negative cells was injected i.p. followed by GCV treatment. Further, survival was prolonged for mice with a preexisting HSV-TK-negative i.p. tumor burden by injecting HSV-TK-positive cells and GCV. These results suggest that genetic modification of tumor cells may be useful for developing cancer therapies.
In photodynamic therapy (PDT), a sensitizer, light, and oxygen are used to cause photochemically induced cell death. The mechanism of cytotoxicity involves generation of singlet oxygen and other free radicals when the light-excited sensitizer loses or accepts an electron. Although selective retention of sensitizer by malignant tissue is seen in vivo, the mechanisms for this sensitizer targeting remain unclear. The first-generation sensitizers are porphyrin based and vary in lipophilicity and hydrophilicity. Targeting of the vasculature seems to be a prominent feature of the cytotoxic effect of these sensitizers in vivo, with resulting necrosis. Treatment depth varies with the wavelength of light that activates the sensitizer used, and the second-generation sensitizers are activated at longer wavelengths, allowing for a 30% increase in treatment depths. The selectivity of targeting can be increased when the sensitizer is delivered with the use of liposomes or monoclonal antibodies specific for tumor antigens. Studies have demonstrated direct effects of PDT on immune effector cells, specifically those with lineage from macrophages or other monocytes. Clinically, this therapy has been chiefly used for palliation of endobronchial and esophageal obstruction, as well as for treatment of bladder carcinomas, skin malignancies, and brain tumors. The future of PDT rests in defining its use either as an intraoperative adjuvant to marginal surgical procedures or as a primary treatment for superficial malignancies. Phase III trials in esophageal cancer and lung cancer are in progress and will help in evaluation of whether Photofrin II, the most widely used sensitizer, can be added to the oncologic armamentarium, pending approval from the U.S. Food and Drug Administration.
Melanin biosynthesis is restricted to melanocytes partly as a consequence of transcriptional regulation of the mRNA coding for those enzymes involved in this biochemical pathway. Promoter sequences of these genes may be used to regulate expression of complementary DNA coding for therapeutic genes so as to provide transcriptional targeting. As a model system we have used the 5'-flanking sequences of the murine tyrosinase or tyrosinase-related protein 1 (TRP-1) genes to show that such transcriptional targeting can be accomplished both in vitro and in vivo. Using interleukin-2 (IL-2) as an example of an immunostimulatory gene and herpes simplex virus thymidine kinase (HSVTK), as an example of a prodrug-activating gene we have shown, in murine model systems, that marked antitumour effects can be achieved by targeted gene therapy approaches. Because other tumor types produce particular proteins as a consequence of specific transcription it is possible that this approach may provide a way of targeting therapeutic genes to various cancers.
The efficiency of 5-aminolevulinic acid (ALA) in photodynamic therapy (PDT) was investigated in vitro using urothelial carcinoma cells of various differentiation. HCV29, RT4 and J82 cells were cultured in 96-well plates, incubated with 25-100 micrograms/ml ALA in serum-containing medium for 4 h, and irradiated at 630, 635 and 640 nm wavelength with light doses of 15-100 J/cm2. The degree of reduced tetrazolium bromide corresponding to cell viability was determined with a colorimetric MTT assay 0, 24 and 48 h after PDT. A remarkable reduction of mitochondrial activity occurred in poorly (J82) and well differentiated (RT4) malignant urothelial cells. Twenty-four hours after photodynamic treatment with 100 micrograms/ml ALA and 50 J/cm2, the metabolic activity of malignant cells was nearly extinguished, while HCV29 cells, derived from normal urothelium, behaved similarly to non-irradiated control cells. The photosensitivity of cells depended on presence or absence of fetal bovine serum (FBS) in the ALA-incubation medium. A wavelength of 635 nm was up to 60% more effective compared with 630 nm, which is more frequently applied in PDT. From the results of our in vitro studies, we can define a "therapeutic window" for malignant cells without damaging benign cells. The time delayed effects and the strong wavelength dependence are important factors for a clinical application.
We are utilizing the folate receptor for the intracellular delivery of DNA. In this study, a folate-poly-L-lysine (FPLL) conjugate was synthesized and equilibrated with plasmid DNA encoding the firefly luciferase gene. The FPLL-DNA complexes were added to KB cells treated with chloroquine. Luciferase activity of cells incubated with FPLL-DNA was 6-fold higher than of cells exposed to poly-L-lysine (PLL)-DNA. The addition of free folic acid competitively inhibited the enhancement of gene expression. Removal of chloroquine from the media significantly inhibited transfection efficiency of FPLL-DNA complexes. We conclude that FPLL-DNA complexes are delivered into KB cells via folate receptor-mediated endocytosis and likely follow a lysosomal pathway into the cytoplasm.
Gene-based therapies for cancer in clinical trials include strategies that involve augmentation of immunotherapeutic and chemotherapeutic approaches. These strategies include ex vivo and in vivo cytokine gene transfer, drug sensitization with genes for prodrug delivery, and the use of drug-resistance genes for bone marrow protection from high-dose chemotherapy. Inactivation of oncogene expression and gene replacement for tumor suppressor genes are among the strategies for targeting the underlying genetic lesions in the cancer cell. A review of clinical trial results to date, primarily in patients with very advanced cancers refractory to conventional treatments, indicates that these treatments can mediate tumor regression with acceptably low toxicity. Vector development remains a critical area for future research. Important areas for future research include modifying viral vectors to reduce toxicity and immunogenicity, increasing the transduction efficiency of nonviral vectors, enhancing vector targeting and specificity, regulating gene expression, and identifying synergies between gene-based agents and other cancer therapeutics.
Safety is an important consideration in the development of genetic therapy protocols; for example, proteins that are therapeutic in the context of one tissue may be harmful in another. This is particularly relevant to suicide gene strategies for cancer, which require in vivo delivery of DNA and which, in general, demand that the therapeutic product be limited as far as possible to malignant cells. This has led to a requirement for "transcriptionally targeted" vectors that can restrict the expression of the therapeutic sequence to appropriate cells. Furthermore, there may be a therapeutic window for certain proteins such that levels of expression below and above certain thresholds may be ineffective or toxic, respectively. Therefore, it would also be desirable to create vectors that allow exogenous control of expression, so that levels of the therapeutic protein can be raised or lowered according to therapeutic need. In the context of transcriptional targeting, one may sometimes use cis-acting sequences to limit transgene expression to the target cell type. In genetic therapy for cancer, for example, it may be possible to identify and use transcriptional control elements that drive expression of proteins unique to, or over-expressed in, malignant cells. These controls would greatly reduce collateral expression of the transgene, and hence reduce toxicity to healthy cells. With regard to exogenous control of expression subsequent to transduction, several synthetic gene regulation systems have now been produced. In these systems, an inducer or repressor acts on a synthetic transcription factor that recognizes motifs unique to the promoter of the transgene; this allows regulated expression of the therapeutic protein without nonspecific effects on cellular promoters. It is likely that a vector will soon be produced in which tissue-restricted expression of the synthetic transcription factor is combined with regulatable transgene expression thereby allowing precise control of therapeutic protein production in specific tissues via administration of an inducing or repressing agent.
In vivo electro-transfection efficiency and manner of transferred gene expression were investigated by fluorescence microscopic image analysis. Green fluorescent protein (GFP) gene was used as the genetic marker. Electroporation was carried out on the liver of live rats by use of disk electrodes mounted in the tips of tweezers, which were directly pressed onto the surface of a liver lobe in situ. Electroporation with eight electric pulses of 50 ms in duration at 50 V gave a good efficiency of transfection as judged by the induced GFP expression. Bright fluorescence of GFP appeared as dots, which were scattered around the area damaged by electroporation. The transfection efficiency increased as the amount of injected DNA was increased. The results indicate that the amount of induced gene expression can be controlled. Estimation of the efficiency of electro-gene transfer using the fluorescence of GFP and digital analysis of microscopic images was useful to determine the optimum conditions for local gene therapy in tissues and organs.
The herpes simplex virus type 1 (HSV-1) virion protein VP22 exhibits the remarkable property of intercellular trafficking whereby the protein spreads from the cell in which it is synthesized to many surrounding cells. In addition to having implications for protein trafficking mechanisms, this function of VP22 might be exploited to overcome a major hurdle in gene therapy, i.e., efficient delivery of genes and gene products. We show that chimeric polypeptides, consisting of VP22 linked to the entire p53 protein, retain their ability to spread between cells and accumulate in recipient cell nuclei. Furthermore the p53-VP22 chimeric protein efficiently induces apoptosis in p53 negative human osteosarcoma cells resulting in a widespread cytotoxic effect. The intercellular delivery of functional p53-VP22 fusion protein is likely to prove beneficial in therapeutic strategies based on restoration of p53 function. These results, demonstrating intracellular transport of large functional proteins, indicate that VP22 delivery may have applications in gene therapy.
Cellular manipulation by transfection or viral introduction of cDNA expression vectors and microinjection of proteins into cells presents various difficulties, including massive overexpression, broad cell-to-cell intracellular concentration ranges of expressed proteins and low percentage of cells targeted1, 2. Moreover, use of antisense approaches to manipulate intracellular processes have both specific gene and cell-type restrictions. Thus, the ability to manipulate cellular processes by the introduction of full-length proteins in a concentration-dependent fashion into 100% of cells would alleviate these technological problems.
Antibody genes of human origin and human antibodies directed against human proteins have become widely available in recent years. These are valuable reagents for gene therapy applications, in which the use of human proteins and genes allows for increased therapeutic benefit. Engineered human antibodies can be used in gene therapy both as a component of a gene delivery system and as a therapeutic gene. As the targeting moiety of a gene delivery system, the antibody should meet certain criteria that have been previously determined from other clinical applications of antibodies. These include bioavailability, specificity for the target cell, and rapid clearance. In addition, if repeat delivery of therapeutic genes is going to be needed, then gene delivery vectors should be non-immunogenic to allow repeated administration. The use of human antibodies in this application should therefore be superior to approaches which use rodent-derived antibodies. Another application of antibodies in gene therapy is the use of antibodies expressed inside the cell (intrabodies) as therapeutic agents. The power of the immune system to rearrange a limited set of genes to create recognition sites for any known molecule is well documented. The ability to harness this information and use these highly specific binding molecules as medicines to inhibit an unwanted cellular function is a promising advance in the field of molecular medicine, and in particular, in the field of intracellular immunization.
The concept behind gene therapy is simple — by delivering corrective genetic material to the cells of a patient the symptoms of disease can be alleviated. But seven years after the first clinical trials on gene therapy began, how far have we come? Gene therapy has not lived up to many of its promises, but the main problem has been in designing efficient delivery vehicles ('vectors'). Nonetheless, the prospects are good — by the year 2010, gene therapy may be as routine a practice as heart transplants are today.