We have investigated the in vivo efficacy of a systemic gene transfer method, which combines a liposomal delivery system (DLS liposomes) Systemic gene therapy by direct delivery of plasmid DNA coupled to synthetic carriers is appealing because of its simplicity, low toxicity, and potential for multiorgan targeting. Although the efficiency of in vitro transfection of DNA plasmids is limited when compared to delivery by viral vectors, recent advances, especially in liposomal delivery, have demonstrated that nonviral gene transfer offers exciting potential (1, 2). Recent attempts to deliver genetic material have provided an impetus to liposome technology (3-6), in particular formation of complexes of DNA with cationic liposomes (7-11). A few therapeutic clinical trial protocols using local administration of these complexes are ongoing but data on systemic administration are still poorly documented (12,13). In this study, we describe a liposomal formulation for gene delivery called DLS, which could be suitable for systemic administration. To obtain long-term expression from a nonintegrated transgene, episomal self-replicating plasmid vectors were constructed and tested. Using the luciferase gene (luc) as a reporter gene (14), we detected transgene expression in various mouse tissues using measurements of luciferase activity, PCR analysis, and immunohistochemistry. Our results demonstrated that a single injection of an episomal DNA vector using the DLS liposome delivery system yielded widespread and long-lasting transgene expression.MATERIALS AND METHODS Materials. Dioctadecylamidoglycylspermidine (7) (15) were deleted to remove expression of these potentially immunogenic proteins. The resultant episomal/reporter expression vector was termed pBKd2RSV-luc. In addition, a second series of pBKdl and pBKd2 vectors were made in which luc expression was placed under the control of the enhancer/promoter sequences from the immediate-early gene of the human cytomegalovirus (CMV) and the polyadenylylation signal and transcriptional termination sequences from the bovine growth hormone gene. The resultant episomal/reporter expression vectors were termed pBKdlCMV-Iuc and pBKd2CMV-Iuc, respectively. The details of these constructs will be reported elsewhere (L.C.M., unpublished data). The nonepisomal pRSV-luc containing luc under control of the RSV long terminal repeat was constructed as reported (14). pCMVintlux plasmid encodes luc under the control of human CMV immediate-early promoter with intron A (16) (generously provided by M. Mansthorpe, Vical, CA). Preparation of the DLS Liposomes. Liposomes were formed by mixing 1 mg of dioctadecylamidoglycylspermidine and 1 mg of dioleoyl phosphatidylethanolamine. After thorough stirring, the mixture was evaporated to dryness in a round-bottomed borosilicate tube using a rotary Vortex evaporator under vacuum. Then the dry lipid film was hydrated with a maximum volume (60 ,ul per mg of lipid) of a solution containing 160 ,ug of plasmid DNA and was slightly Vortex mixed. After in...

We have characterized a new synthetic gene delivery sysplasmid DNA could be detected in blood cells up to 1 h tem, termed DLS, which may be suitable for systemic gene after injection. Systemic administration of DLS-DNA therapy. DLS constitutes a lipopolyamine and a neutral yielded transgene expression in mouse tissues, such as in lipid and associated plasmid DNA in the formation of lamellung or liver. The ratio of DLS:DNA and the procedure used lar vesicles (DLS-DNA). The ratio of lipids and lipid to DNA to form DLS-DNA affected both the level and cellular as well as the method of preparation were optimized to specificity of expression of a luciferase reporter gene yield a high in vitro transfection efficiency compared with showing that in vitro transfection efficiency of DLS-DNA that previously reported for cationic lipid systems. DLSformulations cannot be easily extrapolated to an in vivo set-DNA showed a rapid cellular uptake and distribution in the ting. Optimization of the formulation of a DNA delivery syscytoplasmic and nuclear (especially in the nucleoli) comtem was critical to obtain a defined structure resulting in a partments as determined by laser-assisted confocal preparation with high reproducibility and stability, greater microscopy. There was little or no plasmid DNA degrahomogeneity of particle size and high efficacy following dation over a period of 20 min, relatively slow plasma clearsystemic gene transfer. In addition, the DLS system may ance, and effective and rapid cellular uptake of DLS-DNA be formulated for specific target tissues and may have a following intravenous administration in mice. Supercoiled wide range of applications for gene therapy.Keywords: gene transfer; liposomes; plasmid DNA; biodistribution; pharmacokinetics Synthetic gene transfer vectors are receiving increasingIntroduction study as an alternative to viral vectors since this strategy One of the main impediments to successful gene therapy appears to be safe. Potential methods of gene delivery is the generally poor efficiency of DNA delivery. Most that could be employed include the pneumatic DNA gene therapy efforts involve the use of retroviral vectors gun, 4 DNA-protein complexes 5 or lipidic particles. 6 The because of the usual efficient cell entry and stable intetypical genetic material delivered to target cells by these gration of the gene. Clinical use of retroviral vectors, methods are plasmids, although antisense oligonucleohowever, is hampered by safety issues.1-3 A first concern tides are currently being tested as well. 8 Plasmid prepis the possibility of generating an infectious wild-type arations are simple, quick, safe, inexpensive and may be virus following a recombination event. A second concern applied in combination with a synthetic carrier. Thereis the consequence of random integration of the viral fore, gene therapy by this means may be safe, durable sequence into the genome of the target cell which may and used as a drug-like therapy. The successful use of lead to a tumorigenic or a cytotoxic event. ...

Transfection of human cells with DNA in biomedical applications carries the risk of insertional mutagenesis. Transfection with mRNA avoids this problem; however, in vitro production of mRNA, based on preliminary DNA template cloning in special vectors, is a laborious and time-consuming procedure. We report an efficient vectorfree method of mRNA production from polymerase chain reaction-generated DNA templates. For all cell types tested mRNA was transfected more readily than DNA, and its expression was highly uniform in cell populations. Even cell types relatively resistant to transfection with DNA could express transfected mRNA well. The level of mRNA expression could be controlled over a wide range by changing the amount of input RNA. Cells could be efficiently and simultaneously loaded with several different transcripts. To test a potential clinical application of this method, we transfected human T lymphocytes with mRNA encoding a chimeric immune receptor directed against CD19, a surface antigen widely expressed in leukemia and lymphoma. The transfected mRNA conferred powerful cytotoxicity to T cells against CD19+ targets from the same donor. These results demonstrate that this method can be applied to generate autologous T lymphocytes directed toward malignant cells.

mRNA transfection is a useful approach for temporal cell reprogramming with minimal risk of transgenemediated mutagenesis. We applied this to redirect lymphocyte cytotoxicity toward malignant cells. Using the chimeric immune receptor (CIR) constructs anti-CD19 CIR and 8H9 CIR, we achieved uniform expression of CIRs on virtually the entire population of lymphocytes. We reprogrammed CD3 + CD8 + , CD3 + CD4 + , and natural killer (NK ) cells toward autologous and allogeneic targets such as B cells, Daudi lymphoma, primary melanoma, breast ductal carcinoma, breast adenocarcinoma, and rhabdomyosarcoma. The reprogramming procedure is fast. Although most of the experiments were performed on lymphocytes obtained after 7-day activation, only 1-day activation of T cells with anti-CD3, anti-CD28 antibodies, and interleukin-2 is sufficient to develop both lymphocyte cytotoxicity and competence for mRNA transfer. The entire procedure, which includes lymphocyte activation and reprogramming, can be completed in 2 days. The efficiency of mRNA-modified human T cells was tested in a murine xenograft model. Human CD3 + CD8 + lymphocytes expressing anti-CD19 CIR mRNA inhibited Daudi lymphoma growth in NOD=SCID mice. These results demonstrate that a mixed population of cytotoxic lymphocytes, including T cells together with NK cells, can be quickly and simultaneously reprogrammed by mRNA against autologous malignancies. With relatively minor modifications the described method of lymphocyte reprogramming can be scaled up for cancer therapy.