Frank L. Sorgi

New structures in complex formation between DNA and cationic liposomes visualized by freeze-fracture electron microscopy

Structures formed during interaction of cationic liposomes and plasmid DNA were studied by freeze—fracture electron microscopy and their morphology was found to be dependent on incubation time and DNA concentration. These structures were formed with liposomes composed of DC‐Chol and DOPE after 30 min incubation at DNA: lipid concentrations encompassing maximal transfection activity. They resembled liposome complexes (meatballs) and additionally bilayer‐covered DNA tubules (spaghetti), whereby the DNA‐tubules were found to be connected to the liposome complexes as well as occurring free in the suspension. At later times and higher DNA‐to‐liposome ratios the complexes grow larger while their membranes become discontinuous, allowing the self‐encapsulation of the DNA. The relative transfection potency of the various morphologically distinct structures is discussed.

The tumor suppression activity of E1A in HER-2/neu-overexpressing breast cancer*

The HER-2/neu proto-oncogene is frequently amplified or overexpressed in human breast and ovarian cancers, and is significantly correlated with shorter survival. We have previously reported that the adenovirus type 5 early region 1A (E1A) gene product can repress HER-2/neu overexpression by repressing HER-2/neu promoter activity, and suppress the tumorigenic potential of HER-2/neu-overexpressing ovarian cancer cells. To examine E1A tumor suppressor function in breast cancer, we transduced E1A in vitro by adenovirus into both HER-2/neu-overexpressing and low expressing human breast cancer cell lines. In HER-2/neu-overexpressing cells, E1A greatly inhibited tumor cell growth in vitro. However, in HER-2/neu low expressing cancer cell lines, E1A had no significant effect on cell growth in culture medium. To test the therapeutic efficacy of E1A, we used both adenovirus-mediated and cationic liposome-mediated E1A gene delivery systems in an orthotopic breast cancer animal model. An advanced breast cancer model was established by inoculation of HER-2/neu-overexpressing human breast cancer cells in mammary fat pad and treated by local injections of either replication-deficient adenovirus expressing E1A, Ad.E1A(+) or a liposome-E1A DNA complex. As controls, mice bearing tumors were also treated with Ad.E1A(−) which is virtually the same adenovirus as Ad.E1A(+) except that E1A is deleted, a liposome-E1A frame-shift mutant DNA complex, or just PBS. In mice bearing a HER-2/neu-overexpressing breast cancer cell line, E1A delivered either by adenovirus or liposome significantly inhibited tumor growth and prolonged mouse survival compared with the controls. In fact, 60 – 80% of E1A-treated mice lived longer than 2 years versus only 0 – 20% of control mice (P<0.05). Western blot analysis showed that E1A protein was expressed in tumor tissue and immunohistochemical analysis showed that HER-2/neu p185 protein expression was suppressed. Taken together, our results indicated that both adenovirus and cationic liposome delivery systems were effective in transfering E1A gene for tumor suppression in a HER-2/neu-overexpressing breast cancer model.

HER-2/neu-targeting gene therapy-a review

The HER-2/neu (also named c-erbB-2) oncogene is known to be overexpressed in many human cancers, including breast, ovarian, lung, gastric and oral cancers. In animal models, HER-2/neu overexpression was shown to enhance malignancy and metastasis phenotypes. Repression of HER-2/neu overexpression suppresses the malignant phenotypes of HER-2/neu-overexpressing cancer cells, suggesting that HER-2/neu may serve as an excellent target for developing anti-cancer agents. We have previously shown that the adenovirus-5 (Ad5) E1a gene products and the SV40 large T antigen (large T) inhibit transcription of the HER-2/neu promoter and accordingly suppresses transformation induced by HER-2/neu. In this review, we summarize our recent findings on using cationic liposomes or an Ad vector to deliver E1a or large T into tumor-bearing mice. Our results indicate that both cationic liposomes or an Ad vector can efficiently deliver E1a or large T into tumor cells in mice, and this results in suppression of tumor growth and longer survival of the mice.

Large scale production of DC-Chol cationic liposomes by microfluidization

In this report, we describe the large scale production and testing of DC-Chol cationic liposomes by microfluidization. These liposomes are produced in a GMP acceptable manner to a 500 ml batch size and are shown to be sterile. Further, when stored at 4°C, DC-Chol liposomes will retain their original size, remain suspended in solution, and retain activity for a period exceeding 1.5 years. In-process QA and QC procedures have identified problems in processing and methods to produce a final product of pharmaceutical quality have been developed to overcome these obstacles. Assays for product content (DC-Chol and DOPE assays), size, sterility, endotoxin determination, storage conditions and shelf life have been developed. Successful lots have been used in a human gene therapy clinical trial for cystic fibrosis at Oxford University as well as many pre-clinical experiments throughout the world. Implications for application to further gene therapy clinical trials as well as the development of liposome vector programs are discussed in detail.

DC-Chol lipid system in gene transfer

Abstract Lipidic systems including cationic liposomes offer many potential advantages for delivering functional DNA to cells in intact animals. DC-Chol:DOPE, a cationic liposome formulation developed in this laboratory, is highly efficient for delivering nucleic acids into various cell types in vitro as well as in vivo. Mixing DNA with cationic liposomes produce condensed DNA along with tubular structures and aggregated liposomes. Interaction with cell membrane, followed by endocytosis and disruption of endosomes, appears to be the main mechanism of cytoplasmic delivery of DNA by DNA/cationic liposome complex. In an attempt to overcome the low efficiency of nuclear entry of cytoplasmic DNA, a limiting step for the overall expression level of the transgene, a cytoplasmic expression system was developed. A plasmid containing the reporter gene, chloramphenicol acetyltransferase (CAT), driven by the bacteriophage T7 promoter, following co-delivery with T7 RNA polymerase by DC-Chol cationic liposomes into cells, gives a rapid, but transient CAT gene expression. However, strong and sustained gene expression could be achieved by co-delivery of a T7 RNA polymerase enzyme regenerating system such as a T7 autogene. An independent study found that a single i.p. injection of cisplatin could sensitize lipofection of tumors in situ. A combined and sequential protocol was therefore proposed for cancer gene therapy.

Chapter 12 Drug Delivery Applications

Publisher Summary Liposomes have become an attractive vehicle to target drugs to a specific site. The rationale is to prepare liposomes that are stable and targetable but that will readily destabilize upon reaching the desired target and release their contents. It is assumed that this targeted drug delivery would result in drug being concentrated at the desired location and reduce unwanted toxicity to other areas of the body. One of the principle ingredients in these targetable liposomes is a phospholipid that can exist in a stable bilayer as well as a nonbilayer structure to disrupt the liposome structure. Because of the fine balance between stability and instability of liposomes containing dioleoylphosphatidylethanolamine (DOPE), many researchers have been able to produce a stable liposomal delivery system that can destabilize upon demand. This is done by understanding and controlling the transformation of DOPE from the lamellar phase to the hexagonal phase. Nonbilayer-forming phospholipids have been shown to be excellent candidates for drug delivery and drug targeting. As more research is performed and the destabilizing transitions are better understood and controlled, it can be expected that nonbilayer-forming lipids may play an increasing role in the field of liposome research.

Development of Nonviral DNA Delivery Systems

DNA delivery holds great therapeutic potential, but several barriers have frustrated many creative approaches over the last decade. The presence of an established antiviral immunity in many patients and the rapid induction of an adaptive immune response in naive patients continues to block many attempts to introduce DNA by viral vectors. Nonviral delivery strategies avoid the problems of viral-coat proteins, but encountered new challenges of low and transient expression related to physiological and innate immune barriers. Naked DNA must overcome serum nucleases, conserved immune receptors, nonspecific clearance, cellular membrane barriers, endosomal degradation, and intracellular trafficking to ensure optimal localization and expression (Fig. 1). This chapter reviews the obstacles to nonviral DNA delivery and highlights current formulation strategies designed to ensure efficient localization and expression of therapeutic genes.