Although gene transfer via receptor-targeted polyplexes are mainly successful in delivering DNA efficiently in cell culture, several reports have recently demonstrated the capability of receptor-targeted polyplexes to deliver and transfer DNA to target cells systemically in vivo. This advancement has been enabled by using the various methods to overcome the intra- and extracellular barriers described in the previous sections, but also to a large extent by shielding (or coat) the positive charge of polyplexes, reducing their positive surface charge (zeta potential), hence preventing common interactions with extracellular barriers, such as nonspecific interactions with plasma components or erythrocytes. Shielding methods include those with PEG (153), poly-N-(2-hydroxypropyl)methacrylamide (pHPMA) (165), poloxamer (166), or recently ligand density (23).
A. Receptor-mediated Gene Delivery In Vitro (Ex Vivo)
One approach for performing somatic gene therapy is the ex vivo strategy. In this approach, gene transfer is performed in cell culture (in vitro) and the resulting transfected is transplanted into the organism. Application of receptor-mediated gene transfer to transfect endothelial cells (101,167), fibroblasts, B cells, vessels, and primary tumor cells has potentiated several ex vivo approaches. For example, the clotting factor VIII, which is deficient in hemophilia A, can be produced by transfected primary fibroblasts at levels more than 10-fold higher than those generated by retroviral vectors, enabling factor VIII expressing fibroblast implants for in situ expression of this protein (168).
The highly efficient delivery in vitro has resulted in the development of other ex vivo approaches. For example, treatment for malignant melanoma has been designed by application of gene-modified cancer cell vaccines. DNA complexes are used to deliver immunostimulators genes (e.g., interleukin-2) into melanoma cells in vitro. After irradiation (to block tumor cell growth), the transfected cells are applied in vivo to trigger an antitumor immune response. This treatment has been translated into a medical protocol and is being evaluated in clinical trials (169,170).
The first encouraging results for in vivo gene transfer were obtained by targeting, via intravenous injection, the liver asialog-lycoprotein receptor using asialoorosomucoid covalently linked to poly(L)lysine carrying the CAT marker gene (42-44,46,47). DNA expression proved to be liver specific. Other organs such as kidney, spleen, and lungs did not produce detectable quantities of CAT activity. Gene expression persistence was improved by applying partial hepatectomy, a procedure for stimulation of hepatic regeneration, and DNA synthesis, beginning 12 h after surgery, resulting in CAT activity up to 11 weeks postsurgery.
Related approaches have been used to target the liver, with different size DNA complexes. Unimolecular, about 12-nm small galactosylated DNA-polylysine complexes encoding human factor IX have been shown to target the hepatic asialog-
lycoprotein receptor, with up to 140 days of detectable protein in serum of transfectedrats (60). This result was achieved without partial hepatectomy. In a related approach, the polymeric immunoglobulin receptor has been targeted by 25-nm DNA complexes bearing antigen-binding fragment of an antibody enabling gene transfer to rat pneumocytes following intravenous administration (35,36). It appears that the procedure in preparing DNA complexes may at least partially determine the success of in vivo gene transfer approaches, by influencing extracellular barriers.
Initially, polyplexes based on polylysine did not demonstrate efficient gene transfer in vivo. However, addition of endosomal disruptive agents, such as adenoviruses (84), to such polyplexes resulted in efficient gene expression in airway epithelium after intratracheal application (171) and in tumors after intratumoral application (172,173). Shielding of the polyplexes and the mode of administration, such as with micropump, have further advanced polyplexes to overcome such barriers as extracellular matrix and interstitial pressure (174), respectively. Local injection of DNA complexes directly into subcutane-ously growing tumors produced significant reporter gene expression, with DNA-transferrin-PEI complexes or adenovi-rus-linked DNA-transferrin-polylysine complexes being 10-to 100-fold more efficient than naked DNA (173).
Recently, the lung has been successfully targeted via systemic tail vein injection of miceby 22-kDa linear PEI/DNApol-yplex (175,176). High levels of gene expression were observed in the lungs but lower expression in other major organs, including heart, spleen, kidney, and liver. This observation indicated PEI to be suitable for transfection of the lungs. However, in contrast to the high transfection efficacy observed with PEI in the lungs, systemic gene delivery with PEI/DNA polyplexes to target lung tumors in mice did not result in such an efficient transgene expression (174). Thus, such polyplexes can be classified as nontarget complexes where several parameters may account for the pronounced systemic gene delivery to normal lungs but not to tumors. These include nonspecific interactions of positively charged polyplexes with negatively charged surface structures in the lungs, as well as aggregation of erythro-cytes, resulting in lung embolism and severe toxicity (23).
The promising results discussed above suggest that receptor-targeted polyplexes might beused to deliver genes systemically in vivo to target tumor-specific receptors or receptors that are differentially expressed on tumors (172).
Intravenous application of standard transferrin-PEI-DNA complexes through the tail vein into tumor-bearing mice (subcutaneous tumors) resulted in gene expression in the tail and lung; there was no expression in the tumor, but serious toxicity (153,173). However, surface shielding the transferrin-linked polyplexes with PEG through covalent coupling to PEI, complexes were stabilized in size, did not bind plasma proteins (153) and did not result in erythrocyte aggregation. These PEG-ylated PEI polyplexes, when injected into tail vein of syngeneic mice (see Fig. 5) were far less toxic; gene expression in the tumor was up to 100-fold higher than in other organs (153,173). Similar results were obtained with other transferrin polyplexes with electroneutral surface: shielded optimized adenovirus-linked transferrin-polylysine polyplexes (173) or transferrin-PEI polyplexes with a higher content of transferrin as shielding agent (23).
Because subcutaneous tumors are not directly supplied by main blood vessels, delivery is dependent on peripheral blood supply. Thus, the polyplexes probably target such distant tumors in a combinatorial passive (i.e., overcoming extracellular barriers, EPR = enhanced permeability and retention effect in tumors) and active targeting mechanisms (i.e., specific receptor-targeted endocytosis).
Another recent report demonstrated that intravenous injection of PEGylated EGF-containing DNA-PEI complexes results in a highly specific expression in human hepatocellular carcinoma (HCC) tumors. Following intravenous injection into human HCC xenograft-bearing severe combined immunodeficiency mice, luciferase reporter gene expression was predominantly found in the tumor, with levels up to 2 logs higher than in the liver (52).
Such shielded tumor-targeting polyplexes appear to also have interesting characteristics from a therapeutic perspective. Expression of tumor necrosis factor-a (TNF-a) as the therapeutic gene after systemic application of transferrin- or trans-ferrin-PEG-shielded PEI polyplexes resulted in hemorrhagic necrosis in targeted distant tumors without TNF-a-related systemic toxicity (177,178). These first results are encouraging, and further optimization of tumor-targeting polyplexes should result in the development of potential therapies for human patients.
Was this article helpful?
Complete Guide to Preventing Skin Cancer. We all know enough to fear the name, just as we do the words tumor and malignant. But apart from that, most of us know very little at all about cancer, especially skin cancer in itself. If I were to ask you to tell me about skin cancer right now, what would you say? Apart from the fact that its a cancer on the skin, that is.