- Email: [email protected]
Novel Approaches in Non-Viral Gene Transfer
NOVEL APPROACHES NOVEL APPROACHES 21.
IN
NON-VIRAL GENE TRANSFER
Water Soluble Lipopolymer for Gene Delivery
Sang-oh Han, Ram I. Mahato, Sung Wan Kim Department of Pharmaceuticas and Pharmaceutical Chemistry/CCCD University of Utah, Salt Lake City, UT 84112 Inroduction Gene therapy can be used to deliver cytokine genes into the tumor site, affecting the local tumor environment for the induction of anti-tumor immune response and subsequent tumor eradication. Interleukin-12 (IL-12) has proven to have the most potent antitumor activity. Over the last decade several lipid, peptide and polymer-based vectors have been designed. However, most of these gene carriers show poor in vivo gene expression, high toxicity and poor storage stability. The early success of 3[N, (N’, N’-dimethylaminoethane)-carbaoyl]cholesterol (DC-Chol) lipid-based gene delivery systems spurred recent interest in the development of novel cholesterol-based cationic lipids. However, current cationic lipids are water insoluble and require the formation of cationic liposomes after mixing with a neutral colipid, such as DOPE. For gene transfer, polyethylenimine (PEI) of 25000 Da or above is commonly used, which is highly toxic to the cells even at low N/P ratios. In this study, we designed an effective non-toxic water soluble lipopolymer for cytokine gene delivery. Materials and Methods Water soluble lipopolymer (WSLP) was synthesized using branched PEI of 1800 Da as a cationic headgroup and cholesteryl chloroformate as a hydrophobic lipid anchor. Following synthesis and purification, the structure and molecular weight of WSLP were determined using 400-MHz 1H NMR and MALDI-TOF mass spectrometry. WSLP/pDNA complexes were prepared at different N/P ratios and characterized in terms of particle size, zeta potential, surface morphology, cytotoxicity and in vitro and in vivo gene expression efficiency. Results and Discussion WSLP condensed plasmid DNA when N/P ratio reached to 2.5/1 and no free DNA was detected at N/P ratio 5/1 and above, as determined by agarose gel electrophoresis. The mean particle size was 26 to 62 nm. Atomic force microscopy (AFM) showed complete condensation of plasmid DNA with spherical particles of ⬃50 nm in diameter. WSLP/pDNA complexes were non-toxic to CT-26 colon carcinoma cells when formulated at the N/P ratio of 20/1 and below, as determined by MTT assay. WSLP/p2CMVmIL-12 complexes demonstrated higher transfection efficiency in cultured CT-26 cells (Fig. 1) as well as after intratumoral injection of WSLP/ p2CMVmIL-12 complexes into CT-26 subcutaneous tumor bearing BALB/c mice. WSLP/ p2CMVmIL-12 complexes inhibited tumor progression and prolonged the median survival rate of subcutaneous tumor bearing mice. Conclusion We designed a water soluble non-toxic lipopolymer, which shows enhanced transgene expression both in vitro and in vivo. Acknowledgements We would like to thank Expression Genetics, Inc. for financial support. References S.O. Han, R.I. Mahato and S.W. Kim, Bioconjugate Chem (in press).
MOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
IN
NON-VIRAL GENE TRANSFER
22. High and Persistent Transgene Expression From Plasmid Vectors Containing Hybrid Ubiquitin Promoters Malgorzata Przybylska, Hongmei Zhao, Robin Ziegler, Dapei Liu, Seng H. Cheng, Nelson S. Yew Genzyme Corporation, Framingham, MA 01701 Sustained gene expression will be required for the vast majority of genetic diseases being considered as candidates for gene therapy. Expression from plasmid vectors containing viral promoters, such as that from cytomegalovirus (CMV), is initially high but then declines to near background levels within 2-3 weeks. During our search for cellular promoters that are not inactivated over time in vivo, we have constructed plasmid vectors containing the ubiquitin B (UbB) promoter and evaluated their persistence of expression in the mouse lung. Cationic lipid-plasmid DNA complexes were instilled intranasally (IN) or injected intravenously into immunodeficient BALB/c mice. Chloramphenicol acetyltransferase (CAT) reporter gene expression from the UbB constructs was initially very low at day 2 post-administration, but at day 35 exceeded the level of expression from the CMV promoter vector by 4-9 fold. Appending a portion of the CMV enhancer to the 5⬘ end of the UbB promoter increased CAT expression at early time points to nearly that of the CMV promoter vector, and expression was persistent for at least three months, with 50% of day 2 levels remaining at day 84. Expression from the CMVubiquitin promoter vector was also sustained for 42 days in the mouse liver. Since previous studies have shown that eliminating immunostimulatory CpG motifs in plasmid DNA vectors reduces their toxicity, we have also constructed a CpG deficient version of the CMV-UbB vector (pGZCUBI-HAGA) encoding an ␣-galactosidase transgene, the enzyme that is deficient in Fabry disease, a lysosomal storage disorder (LSD). After IN administration, ␣-galactosidase expression from pGZCUBI-HAGA rose steadily over time and reached levels 25 fold higher than from a CMV promoter vector at day 35. Current efforts are to evaluate the persistence of pGZCUBI vectors after systemic delivery and their applicability to treating other LSDs as well as hemophilias. These results suggest that CpG-reduced plasmid vectors containing CMV-ubiquitin promoter hybrids may provide the greater persistence and efficacy required for a practical gene therapeutic.
23. Selective brain cell expression with non-viral gene delivery vectors Arron Hirko*†‡, Mike King*†‡, Ronald Kline*†‡, Ed Meyer*†‡, Jeffrey Hughes*†‡ *University of Florida †Center for Neurobiology of Aging ‡Gainesville Fl 32610 Safe and efficient gene delivery systems are critical factors for successful brain gene therapy. Progress has been made in improving transfection efficiency of cationic lipid-plasmid systems in vivo. Problems remaining include toxicity of some vectors, shortterm duration of expression and low numbers of cells transfected. We have synthesized new cationic lipids that differ relative to intrinsic chemical properties, predicated interactions with plasmids and intracellular disposition. Several of these lipids appear to be more effective with less accompanying toxicity for plasmid expression in the rat brain than known non-viral agents, at least when using adeno associated virus (AAV) derived plasmids. The AAV-derived plasmid used in these experiments, and the AAV derived vector, contained identical AAV sequences, and terminal repeats. Both gene transfer systems also used the chicken beta actin (CBA) promoter to drive GFP expression directly. In addition, the CBA promoter also drives expression in glia, making it possible to answer additivity questions. The three non-viral transfer vehicles used were a synthetic commercial obtainable cationic polymer PEI that has demonstrable effects in brain tissues, CHDTAEA, a cholesterol based disulfide lipid, and a single tailed surfactant with an ester in the backbone(OLON). In both of the surface-active cases the liposomes were prepared with equal
S9
NOVEL APPROACHES
IN
NON-VIRAL GENE TRANSFER
molar ratio of DOPE. These substances varied in their activities in vitro relative to toxicity and pattern of glial/neuronal transfection, as noted above, but each drove at least some expression in brain. Figure 1 illustrates the expression of GFP tested via immunostaining with the tested delivery systems. Interestingly, we found at least one type of lipid transfection agent permits transfection of glia but low neuron transfection in vivo (CHDTAEA), while other non-viral transfection agents give high levels of transfection in neurons and glia cells. For these initial in vivo studies, we injected AAV viral vectors as positive controls into one side of the brain and various treatments contralaterally (vehicle only; vehicle ⫹ plasmid; vehicle⫹plasmid⫹ viral vector; vehicle ⫹ viral vector). This positive-control, contralateral paradigm provided reassurance that the immunohistochemical and injection procedures were effective and reproducible in each animal. In a separate set of animals a similar injection scheme was conducted but wet brain section were isolated and mRNA content was determined via RT-PCR. The results indicate that AAV-derived non-viral gene transfer is likely to be sufficiently robust alone, and under some conditions additive with AAV viral vectors, to permit testing of hypotheses regarding non-viral somatic gene transfer and trophic factor efficacy in brain. The amount of expression observed with rAAV alone in this study is comparable to previously reported results with this vector system in hippocampus and septum. The additive expression observed with rAAV and plasmid AAV vectors may lead to even greater transgene expression.
24. Bihead Lipids: a new class of cationic lipids as gene delivery carrier Jingping Yang*, Alexander Chanturiya*, Puthupparampil Scaria*§, Jinghai Wang*, chaoning Gu*, Helen Awatefe*, ke Weng*, Yangping Pan*, Richard Titmas*, Jaroslav Stanek†, Giorgio Caravatti†, Marc Lang†, Joerg Frei†, Helmut Mett§, Donna B. Stolz‡, Simon C. Watkins‡, Woodle C. Martin*§ *Genetic Therapy Inc., a Novartis Company, Gaithersburg, MD20878 †Novartis Pharma AG, Basel, Switzland ‡Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, PA15261 §Current address: Intradigm Corporation, 8205 Beech Tree Road Bethesda, MD 20817 Lipofection has been intensively used in biological research and studied in gene therapy clinical trials. Currently published cationic lipids have only one polar head and one hydrophobic moiety and thus form bilayer membrane. After investigating a series of polyamine analogues as gene delivery vehicles and analyzing their structure and activity relationship, the present study proposes a new class of cationic lipids, bihead lipids. Bihead lipids have two positively charged polar heads connected by one hydrophobic carbon chain. Each head extends one hydrophobic carbon chain that, like a hand, tend to enclose the central chain by Van del Waals attractive force. The three carbon chains form one hydrophobic body. The dispersion of bihead lipids in water formed micelles with a diameter of 5-30 nm. Their bihead structure suggests that they may form monolayer membranes in water. The bihead lipids showed relatively high transfection activity in vitro and in vivo with up to 86% of primary synovium from arthritis patient transfected. The lipoplexes of bihead lipids and plasmids were characterized by particle size and electron microscopy. The interaction of two particular bihead lipids with planar bilayer membranes was studied. They form relatively stable, large, and anion-selective pores. Furthermore, one particular bihead lipid was modified by conjugation with PEG5000 to one head via a cleaveable disulfied bond. Modification of one head did not affect the other unmodified head binding with DNA, but the transfection activity was lost. Addition of DTT to release PEG5000 recovered the transfection activity of the modified bihead lipid. The applications of bihead lipids as gene and drug delivery vehicles is discussed.
S10
25. CATIONIC LIPID POLYMERIZATION AS A NOVEL APPROACH TO IMPROVE LIPOPLEX STABILITY, TOXICITY AND TRANSFECTION EFFICIENCY Jian Wu*, Mike E. Lizarzaburu†, Li Liu*, Henning Wege*, Mark J. Kurth†, Mark A. Zern*, Michael H. Nantz† *Transplant Research Institute, UC Davis Medical Center, Sacramento, CA 95817 †Department of Chemistry, UC Davis, Davis, CA 95616 Background and aims: In vivo gene delivery mediated by cationic lipids is often compromised by the formation of large aggregations due to proteins in the blood. In order to improve the stability of cationic lipids, the present study aims to develop a novel approach in which an acrylamide cationic lipid is polymerized so that the resultant lipidic poly(acrylamide) (LPA) yields stable cationic liposomes for gene therapy. To test this a monomeric hydrogenated lipid (MHL) of the polymer was utilized for comparison. Methods: The acrylamide lipid was synthesized from 3-bromo-1,2-propanediol, and polymerized by specific chemical treatment of its aqueous suspension. Various formulations of cationic liposomes, such as MHL-cholesterol (Ch), LPADOPE, LPA-Ch, DOTAP-DOPE and DOTAP-Ch at 1:1 molar ratio were generated by the hydration of dried lipid mixtures and brief sonication. Liposome-mediated transfection of a vector (pGL3), which encodes a luciferase reporter gene, was evaluated in Hep G2 and Alexander cell lines. Results: After the liposomes were dispersed in the medium containing 10% FBS, the size of MHL, MHL-Ch, and DOTAP-Ch was markedly increased (180⫾66, 185⫾61, 92⫾15 vs. 563⫾240, 512⫾170, 996⫾83 nm), but the size of LPA, LPA-Ch was only slightly increased (284⫾50, 269⫾48 vs. 354⫾140, 316⫾130 nm). The size of LPA remained largely the same (from 377⫾76 to 458⫾72 nm) when LPA was added into MEM medium containing 10, 50 and 100% serum for 24 hours, whereas, it changed extensively for MHL (from 180⫾69 to 1836⫾299 nm), and so did Lipofectamine in 50% serum. LPA at 20 microgram/ml did not display any significant toxicity to rat hepatocytes and less toxicity at a higher concentration (40 microgram/ml) to Hep G2 cells when compared to MHL (p⬍0.01). When LPA formed liposomes with Ch, its toxicity to rat hepatocytes was lower than Lipofectamine (4.5⫾0.4 vs. 11.0⫾3.0%, p⬍0.01). LPA-Ch and LPA-DOPE led to luciferase activity 5-7.5 fold higher (2.7X10E7, 3.5X10E7 RLU) in Hep G2 cells than LPA alone (4.7X10E6, 6.5X10E6 RLU). The activity was also considerably higher (5 fold) than liposomes made from the non-polymerized lipids (MHL-Ch). Moreover, the activity of the polymerized liposomes (LPA-Ch) was as high as Lipofectamine when it was used as a standard transfecting agent. Conclusions: Polymerized liposome formulations are more stable in medium containing various concentrations of serum, are less toxic, and exhibit better transfection when they are incorporated with a co-lipid, such as cholesterol. Their transfection efficiency matches that of Lipofectamine. Thus, this novel approach for the development of stable and active polyplexes may prove to be a valuable alternative for in vivo gene delivery.
26. Efficient Systemic Delivery of Factor VIII Gene by Poly(ethylene glycol)-Grafted, Galactose-Conjugated Neutral-Cationic Lipid/DNA Complex SK Huang, B. Jin, W. Zhang, N. Mullah, S. Zalipsky ALZA Corporation, 1900 Charleston Road, Mountain View, CA 94039-7210 Systemic administration of cationic liposome/DNA complexes leads to their facile entrapment in the lung. This lung localization is caused by the strong positive surface charge of the complexes, which is also important for DNA complexation, cell interaction, and DNA transfer from the lysosomes to the cytoplasm after internalization. It has been a formidable challenge, however, to change the DNA-complex distribution from the lung to other desired tissues, while at the same time preserving transfection efficiency. In this study, we synthesized a new pH-depenMOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
NOVEL APPROACHES dent, neutral-cationic lipid (NCL), histamine distearoyl glycerol, and tested whether its use in the liposomal complex, together with PEG and ligand surface modifications, would improve distribution beyond the lung while maintaining transfection efficiency. Histamine distearoyl glycerol tends to be neutral at physiological pH (imidazole of histamine has a pKa of 6). The absence of surface charge at physiological pH is critical for increasing blood retention time and changing tissue distribution of these complexes following systemic administration. Histamine distearoyl glycerol is predominantly positively charged at pH ⬍ 6, which may facilitate the interaction of the complexes with the lysosomal membrane and ultimately the release of the DNA content into the cytoplasm. Complexes were prepared with liposomes composed of NCL/PHSPC (partially hydrogenated soybean phosphatidylcholine) at 40:60 molar ratio and plasmid DNA encoding human Factor VIII gene (B-domain deleted). The surface of the complexes was modified with 10 mol% mPEG2000DSPE and approximately 20 galactose-poly(ethylene glycol)-distearoylphosphatidic acid molecules per complex. These complexes were injected intravenously to C57BL6 mice (100 g DNA in 0.4 mL), and the plasma concentration of expressed human Factor VIII was determined 24 hours after treatment using the human Factor VIII Coatest assay. Our results show that NCLliposomes encapsulated DNA and formed complexes at pH 4 to 5. The NCL-liposome complexes were stable at pH 7.4, as confirmed by DNase and pico-green assays. By zeta-potential measurements, the surface potential of these complexes was 25 mV at pH 5 and 0 mV at pH 7.4. In a tissue distribution study, the accumulation of NCL liposome/DNA complexes demonstrated around 10-fold higher in the liver as compared to the lung after intravenous injection. After treatment with the new complex, the mean plasma level of Factor VIII was 4.7 (⫾2.8) ng/mL. Only 0.86 (⫾0.3) ng/mL of Factor VIII could be detected after treatment with complexes without PEG and ligand surface modifications. No Factor VIII expression was detected with the conventional DDAB cationic liposome/DNA complexes under the same conditions. Administration of 100 g of naked plasmid DNA by 1-mL bolus injection volume and 0.2-mL small injection volume achieved plasma concentrations of Factor VIII of 1.2 (⫾0.3) ng/mL and 0.80 (⫾1.3) ng/mL, respectively. The results of this study indicate that the use of the NCL liposome/DNA complexes with specific surface modifications improves delivery to the desired tissues, facilitates internalization into target cells, and provides a useful approach for the systemic delivery of clinically relevant genes. Acknowledgements: The human Factor VIII plasmid and Factor VIII coatest assay were provided by Dr. Phil Scuderi’s group, Bayer Corporation.
27. Targeting of Lipid-Protamine-DNA (LPD) complexes using DSPE-PEG5000-LHRH or DSPEPEG5000-RGD Pierrot Harvie*, Ben Dutzar*, Todd Galbraith*, Sally Cudmore†, Dan O’Mahony†, Pervin Ankleseria*, Ralph Paul* *Targeted Genetics Corporation, 1100 Olive Way Seattle, WA 98101 USA †Elan Pharmaceutical Technologies, Dublin, Ireland Lipid-Protamine-DNA (LPD) lipopolyplexes have successfully been used for systemic gene transfer (Li et al. (1998) Gene Therapy, 5: 930-937). The use of polyethylene glycol (PEG)-modified lipids is well established for liposome encapsulated drugs and their ability to enhance the delivery of anti-cancer drugs to tumor sites has been proven (Lasic, (1998) Trends in Biotechnology 16; 307-32). Pegylated lipids are also known to control surface properties of cationic lipid-based gene transfer systems. However, pegylated lipid incorporation into lipid-DNA complexes causes a concentration dependent reduction of in vitro transfection activity, a result that can be partially attributed to a reduction in particle binding to cells (Harvie et al. J. Phar Sci. (2000) 89;5 652-663). In order to restore particle binding MOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
IN
NON-VIRAL GENE TRANSFER
and target LPD to tumor cell, the lipid-peptide conjugates DSPE-PEG5k-LHRH (Leutinizing Hormone Release Hormone) or DSPE-PEG5k-RGD were generated and incorporated into a lipid-protamine-DNA (LPD) formulation. Addition of such lipidconjugated-ligands are compatible with LPD formation. Lipidconjugated-ligand incorporation into LPDs results in a dosedependent enhancement of LPD formulation binding to cells as well as a corresponding increase in luciferase expression following in vitro transfection of MDA-MB-231 and SKOV-3 cells. Maximum luciferase expression was observed at 10 mol percent lipidconjugated-ligand incorporation into the LPD formulations. Addition of 10 mol percent of DSPE-PEG5k-LHRH to an LPD formulation enhances transfection activity in vitro in MDA-MB231 cells by 285 fold over the base pegylated LPD formulation. DSPE-PEG5k-RGD incorporation into an LPD resulted in a transfection activity enhancement in vitro in MDA-MB-231 cells up to 100 fold. Moreover, transfection activity of LPD bearing DSPEPEG5k-LHRH can be abolished in a competition assay using 1000 fold excess of free LHRH. DSPE-PEG5k-RGD mediated transfection enhancement can also be abolished in a competition assay using 1000 fold excess of free RGD peptide, while an RGE control peptide had no effect. These data suggest that this those new systemic gene delivery formulations are extremely versatile for controlling LPD behavior and will lead to more effective cell specific targeting. This work was supported by Emerald Gene systems
28. Lipid/mu/DNA Complexes as Gene Transfer Agents M. Manvell*, T. Tagawa†, E. Perouzel†, A. Miller†, EWFW Alton* *Deptartment of Gene Therapy, Imperial College School of Medicine at the National Heart and Lung Institute, London, England †Imperial College Genetic Therapies Centre, Department of Chemistry, Imperial College, London, England For in vivo systemic gene transfer, both the volume in which DNA:lipid complexes are delivered and the potentially toxic effects of either component limit gene expression. We have assessed whether addition of the adenoviral mu protein to cationic lipids allows for more efficient gene transfer. Lipid/mu/DNA complexes (LMDs) were used to transfect COS 7 cells, with the ratio of Lipid:mu:DNA remaining constant at 12:0.6:1, whilst DNA and hence mu and lipid doses were varied. At the highest dose of DNA (5 g), standard DNA-DC-Cholesterol:DOPE (DCChol) lipid complexes (at 2:1 lipid:DNA) produced significantly (p⬍0.05, n⫽14) greater expression than equivalent doses of LMDs. However, at the lower dose of 0.5 g DNA the LMD complex performed significantly (p⬍0.01, n⫽8) better than DCChol with values equal to the transfection level seen with DCChol at 5 g DNA. When a time course was performed, LMD showed significant gene expression (assayed 48 hours later) after only 30 minutes incubation with the transfection mixture, whereas the DC-Chol complex only showed significant expression after 24 hours incubation. In the presence of 10% serum transfection efficiency was significantly (p⬍0.01, n⫽10) improved using LMDs (5 g DNA). In contrast, DC-Chol-mediated transfection was markedly reduced (p⬍0.01, n⫽10). 100% serum (5 g DNA) significantly (p⬍0.05, n⫽6) reduced LMD transfections compared to no serum but gene transfer could still be clearly seen. In contrast DC-Chol in the presence of 100% serum (at 2:1 lipid:DNA dose) showed no evidence of gene transfer. However, the 12:1 DC-Chol/DOPE:DNA control complexes performed as well as LMD in 100% serum, suggesting that the excess lipid in the LMD complex is responsible for the protective effect in serum. In conclusion, in vitro LMDs produced the same transfection efficiency as DC-Chol/DOPE complexes with one log order less DNA, and showed transfection at a much earlier time point. LMD could also produce gene transfer in the presence of 100% serum, due to the presence of excess lipid. These properties may make LMDs suitable for systemic delivery applications.
S11
IN
NON-VIRAL GENE TRANSFER
Water Soluble Lipopolymer for Gene Delivery
Sang-oh Han, Ram I. Mahato, Sung Wan Kim Department of Pharmaceuticas and Pharmaceutical Chemistry/CCCD University of Utah, Salt Lake City, UT 84112 Inroduction Gene therapy can be used to deliver cytokine genes into the tumor site, affecting the local tumor environment for the induction of anti-tumor immune response and subsequent tumor eradication. Interleukin-12 (IL-12) has proven to have the most potent antitumor activity. Over the last decade several lipid, peptide and polymer-based vectors have been designed. However, most of these gene carriers show poor in vivo gene expression, high toxicity and poor storage stability. The early success of 3[N, (N’, N’-dimethylaminoethane)-carbaoyl]cholesterol (DC-Chol) lipid-based gene delivery systems spurred recent interest in the development of novel cholesterol-based cationic lipids. However, current cationic lipids are water insoluble and require the formation of cationic liposomes after mixing with a neutral colipid, such as DOPE. For gene transfer, polyethylenimine (PEI) of 25000 Da or above is commonly used, which is highly toxic to the cells even at low N/P ratios. In this study, we designed an effective non-toxic water soluble lipopolymer for cytokine gene delivery. Materials and Methods Water soluble lipopolymer (WSLP) was synthesized using branched PEI of 1800 Da as a cationic headgroup and cholesteryl chloroformate as a hydrophobic lipid anchor. Following synthesis and purification, the structure and molecular weight of WSLP were determined using 400-MHz 1H NMR and MALDI-TOF mass spectrometry. WSLP/pDNA complexes were prepared at different N/P ratios and characterized in terms of particle size, zeta potential, surface morphology, cytotoxicity and in vitro and in vivo gene expression efficiency. Results and Discussion WSLP condensed plasmid DNA when N/P ratio reached to 2.5/1 and no free DNA was detected at N/P ratio 5/1 and above, as determined by agarose gel electrophoresis. The mean particle size was 26 to 62 nm. Atomic force microscopy (AFM) showed complete condensation of plasmid DNA with spherical particles of ⬃50 nm in diameter. WSLP/pDNA complexes were non-toxic to CT-26 colon carcinoma cells when formulated at the N/P ratio of 20/1 and below, as determined by MTT assay. WSLP/p2CMVmIL-12 complexes demonstrated higher transfection efficiency in cultured CT-26 cells (Fig. 1) as well as after intratumoral injection of WSLP/ p2CMVmIL-12 complexes into CT-26 subcutaneous tumor bearing BALB/c mice. WSLP/ p2CMVmIL-12 complexes inhibited tumor progression and prolonged the median survival rate of subcutaneous tumor bearing mice. Conclusion We designed a water soluble non-toxic lipopolymer, which shows enhanced transgene expression both in vitro and in vivo. Acknowledgements We would like to thank Expression Genetics, Inc. for financial support. References S.O. Han, R.I. Mahato and S.W. Kim, Bioconjugate Chem (in press).
MOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
IN
NON-VIRAL GENE TRANSFER
22. High and Persistent Transgene Expression From Plasmid Vectors Containing Hybrid Ubiquitin Promoters Malgorzata Przybylska, Hongmei Zhao, Robin Ziegler, Dapei Liu, Seng H. Cheng, Nelson S. Yew Genzyme Corporation, Framingham, MA 01701 Sustained gene expression will be required for the vast majority of genetic diseases being considered as candidates for gene therapy. Expression from plasmid vectors containing viral promoters, such as that from cytomegalovirus (CMV), is initially high but then declines to near background levels within 2-3 weeks. During our search for cellular promoters that are not inactivated over time in vivo, we have constructed plasmid vectors containing the ubiquitin B (UbB) promoter and evaluated their persistence of expression in the mouse lung. Cationic lipid-plasmid DNA complexes were instilled intranasally (IN) or injected intravenously into immunodeficient BALB/c mice. Chloramphenicol acetyltransferase (CAT) reporter gene expression from the UbB constructs was initially very low at day 2 post-administration, but at day 35 exceeded the level of expression from the CMV promoter vector by 4-9 fold. Appending a portion of the CMV enhancer to the 5⬘ end of the UbB promoter increased CAT expression at early time points to nearly that of the CMV promoter vector, and expression was persistent for at least three months, with 50% of day 2 levels remaining at day 84. Expression from the CMVubiquitin promoter vector was also sustained for 42 days in the mouse liver. Since previous studies have shown that eliminating immunostimulatory CpG motifs in plasmid DNA vectors reduces their toxicity, we have also constructed a CpG deficient version of the CMV-UbB vector (pGZCUBI-HAGA) encoding an ␣-galactosidase transgene, the enzyme that is deficient in Fabry disease, a lysosomal storage disorder (LSD). After IN administration, ␣-galactosidase expression from pGZCUBI-HAGA rose steadily over time and reached levels 25 fold higher than from a CMV promoter vector at day 35. Current efforts are to evaluate the persistence of pGZCUBI vectors after systemic delivery and their applicability to treating other LSDs as well as hemophilias. These results suggest that CpG-reduced plasmid vectors containing CMV-ubiquitin promoter hybrids may provide the greater persistence and efficacy required for a practical gene therapeutic.
23. Selective brain cell expression with non-viral gene delivery vectors Arron Hirko*†‡, Mike King*†‡, Ronald Kline*†‡, Ed Meyer*†‡, Jeffrey Hughes*†‡ *University of Florida †Center for Neurobiology of Aging ‡Gainesville Fl 32610 Safe and efficient gene delivery systems are critical factors for successful brain gene therapy. Progress has been made in improving transfection efficiency of cationic lipid-plasmid systems in vivo. Problems remaining include toxicity of some vectors, shortterm duration of expression and low numbers of cells transfected. We have synthesized new cationic lipids that differ relative to intrinsic chemical properties, predicated interactions with plasmids and intracellular disposition. Several of these lipids appear to be more effective with less accompanying toxicity for plasmid expression in the rat brain than known non-viral agents, at least when using adeno associated virus (AAV) derived plasmids. The AAV-derived plasmid used in these experiments, and the AAV derived vector, contained identical AAV sequences, and terminal repeats. Both gene transfer systems also used the chicken beta actin (CBA) promoter to drive GFP expression directly. In addition, the CBA promoter also drives expression in glia, making it possible to answer additivity questions. The three non-viral transfer vehicles used were a synthetic commercial obtainable cationic polymer PEI that has demonstrable effects in brain tissues, CHDTAEA, a cholesterol based disulfide lipid, and a single tailed surfactant with an ester in the backbone(OLON). In both of the surface-active cases the liposomes were prepared with equal
S9
NOVEL APPROACHES
IN
NON-VIRAL GENE TRANSFER
molar ratio of DOPE. These substances varied in their activities in vitro relative to toxicity and pattern of glial/neuronal transfection, as noted above, but each drove at least some expression in brain. Figure 1 illustrates the expression of GFP tested via immunostaining with the tested delivery systems. Interestingly, we found at least one type of lipid transfection agent permits transfection of glia but low neuron transfection in vivo (CHDTAEA), while other non-viral transfection agents give high levels of transfection in neurons and glia cells. For these initial in vivo studies, we injected AAV viral vectors as positive controls into one side of the brain and various treatments contralaterally (vehicle only; vehicle ⫹ plasmid; vehicle⫹plasmid⫹ viral vector; vehicle ⫹ viral vector). This positive-control, contralateral paradigm provided reassurance that the immunohistochemical and injection procedures were effective and reproducible in each animal. In a separate set of animals a similar injection scheme was conducted but wet brain section were isolated and mRNA content was determined via RT-PCR. The results indicate that AAV-derived non-viral gene transfer is likely to be sufficiently robust alone, and under some conditions additive with AAV viral vectors, to permit testing of hypotheses regarding non-viral somatic gene transfer and trophic factor efficacy in brain. The amount of expression observed with rAAV alone in this study is comparable to previously reported results with this vector system in hippocampus and septum. The additive expression observed with rAAV and plasmid AAV vectors may lead to even greater transgene expression.
24. Bihead Lipids: a new class of cationic lipids as gene delivery carrier Jingping Yang*, Alexander Chanturiya*, Puthupparampil Scaria*§, Jinghai Wang*, chaoning Gu*, Helen Awatefe*, ke Weng*, Yangping Pan*, Richard Titmas*, Jaroslav Stanek†, Giorgio Caravatti†, Marc Lang†, Joerg Frei†, Helmut Mett§, Donna B. Stolz‡, Simon C. Watkins‡, Woodle C. Martin*§ *Genetic Therapy Inc., a Novartis Company, Gaithersburg, MD20878 †Novartis Pharma AG, Basel, Switzland ‡Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, PA15261 §Current address: Intradigm Corporation, 8205 Beech Tree Road Bethesda, MD 20817 Lipofection has been intensively used in biological research and studied in gene therapy clinical trials. Currently published cationic lipids have only one polar head and one hydrophobic moiety and thus form bilayer membrane. After investigating a series of polyamine analogues as gene delivery vehicles and analyzing their structure and activity relationship, the present study proposes a new class of cationic lipids, bihead lipids. Bihead lipids have two positively charged polar heads connected by one hydrophobic carbon chain. Each head extends one hydrophobic carbon chain that, like a hand, tend to enclose the central chain by Van del Waals attractive force. The three carbon chains form one hydrophobic body. The dispersion of bihead lipids in water formed micelles with a diameter of 5-30 nm. Their bihead structure suggests that they may form monolayer membranes in water. The bihead lipids showed relatively high transfection activity in vitro and in vivo with up to 86% of primary synovium from arthritis patient transfected. The lipoplexes of bihead lipids and plasmids were characterized by particle size and electron microscopy. The interaction of two particular bihead lipids with planar bilayer membranes was studied. They form relatively stable, large, and anion-selective pores. Furthermore, one particular bihead lipid was modified by conjugation with PEG5000 to one head via a cleaveable disulfied bond. Modification of one head did not affect the other unmodified head binding with DNA, but the transfection activity was lost. Addition of DTT to release PEG5000 recovered the transfection activity of the modified bihead lipid. The applications of bihead lipids as gene and drug delivery vehicles is discussed.
S10
25. CATIONIC LIPID POLYMERIZATION AS A NOVEL APPROACH TO IMPROVE LIPOPLEX STABILITY, TOXICITY AND TRANSFECTION EFFICIENCY Jian Wu*, Mike E. Lizarzaburu†, Li Liu*, Henning Wege*, Mark J. Kurth†, Mark A. Zern*, Michael H. Nantz† *Transplant Research Institute, UC Davis Medical Center, Sacramento, CA 95817 †Department of Chemistry, UC Davis, Davis, CA 95616 Background and aims: In vivo gene delivery mediated by cationic lipids is often compromised by the formation of large aggregations due to proteins in the blood. In order to improve the stability of cationic lipids, the present study aims to develop a novel approach in which an acrylamide cationic lipid is polymerized so that the resultant lipidic poly(acrylamide) (LPA) yields stable cationic liposomes for gene therapy. To test this a monomeric hydrogenated lipid (MHL) of the polymer was utilized for comparison. Methods: The acrylamide lipid was synthesized from 3-bromo-1,2-propanediol, and polymerized by specific chemical treatment of its aqueous suspension. Various formulations of cationic liposomes, such as MHL-cholesterol (Ch), LPADOPE, LPA-Ch, DOTAP-DOPE and DOTAP-Ch at 1:1 molar ratio were generated by the hydration of dried lipid mixtures and brief sonication. Liposome-mediated transfection of a vector (pGL3), which encodes a luciferase reporter gene, was evaluated in Hep G2 and Alexander cell lines. Results: After the liposomes were dispersed in the medium containing 10% FBS, the size of MHL, MHL-Ch, and DOTAP-Ch was markedly increased (180⫾66, 185⫾61, 92⫾15 vs. 563⫾240, 512⫾170, 996⫾83 nm), but the size of LPA, LPA-Ch was only slightly increased (284⫾50, 269⫾48 vs. 354⫾140, 316⫾130 nm). The size of LPA remained largely the same (from 377⫾76 to 458⫾72 nm) when LPA was added into MEM medium containing 10, 50 and 100% serum for 24 hours, whereas, it changed extensively for MHL (from 180⫾69 to 1836⫾299 nm), and so did Lipofectamine in 50% serum. LPA at 20 microgram/ml did not display any significant toxicity to rat hepatocytes and less toxicity at a higher concentration (40 microgram/ml) to Hep G2 cells when compared to MHL (p⬍0.01). When LPA formed liposomes with Ch, its toxicity to rat hepatocytes was lower than Lipofectamine (4.5⫾0.4 vs. 11.0⫾3.0%, p⬍0.01). LPA-Ch and LPA-DOPE led to luciferase activity 5-7.5 fold higher (2.7X10E7, 3.5X10E7 RLU) in Hep G2 cells than LPA alone (4.7X10E6, 6.5X10E6 RLU). The activity was also considerably higher (5 fold) than liposomes made from the non-polymerized lipids (MHL-Ch). Moreover, the activity of the polymerized liposomes (LPA-Ch) was as high as Lipofectamine when it was used as a standard transfecting agent. Conclusions: Polymerized liposome formulations are more stable in medium containing various concentrations of serum, are less toxic, and exhibit better transfection when they are incorporated with a co-lipid, such as cholesterol. Their transfection efficiency matches that of Lipofectamine. Thus, this novel approach for the development of stable and active polyplexes may prove to be a valuable alternative for in vivo gene delivery.
26. Efficient Systemic Delivery of Factor VIII Gene by Poly(ethylene glycol)-Grafted, Galactose-Conjugated Neutral-Cationic Lipid/DNA Complex SK Huang, B. Jin, W. Zhang, N. Mullah, S. Zalipsky ALZA Corporation, 1900 Charleston Road, Mountain View, CA 94039-7210 Systemic administration of cationic liposome/DNA complexes leads to their facile entrapment in the lung. This lung localization is caused by the strong positive surface charge of the complexes, which is also important for DNA complexation, cell interaction, and DNA transfer from the lysosomes to the cytoplasm after internalization. It has been a formidable challenge, however, to change the DNA-complex distribution from the lung to other desired tissues, while at the same time preserving transfection efficiency. In this study, we synthesized a new pH-depenMOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
NOVEL APPROACHES dent, neutral-cationic lipid (NCL), histamine distearoyl glycerol, and tested whether its use in the liposomal complex, together with PEG and ligand surface modifications, would improve distribution beyond the lung while maintaining transfection efficiency. Histamine distearoyl glycerol tends to be neutral at physiological pH (imidazole of histamine has a pKa of 6). The absence of surface charge at physiological pH is critical for increasing blood retention time and changing tissue distribution of these complexes following systemic administration. Histamine distearoyl glycerol is predominantly positively charged at pH ⬍ 6, which may facilitate the interaction of the complexes with the lysosomal membrane and ultimately the release of the DNA content into the cytoplasm. Complexes were prepared with liposomes composed of NCL/PHSPC (partially hydrogenated soybean phosphatidylcholine) at 40:60 molar ratio and plasmid DNA encoding human Factor VIII gene (B-domain deleted). The surface of the complexes was modified with 10 mol% mPEG2000DSPE and approximately 20 galactose-poly(ethylene glycol)-distearoylphosphatidic acid molecules per complex. These complexes were injected intravenously to C57BL6 mice (100 g DNA in 0.4 mL), and the plasma concentration of expressed human Factor VIII was determined 24 hours after treatment using the human Factor VIII Coatest assay. Our results show that NCLliposomes encapsulated DNA and formed complexes at pH 4 to 5. The NCL-liposome complexes were stable at pH 7.4, as confirmed by DNase and pico-green assays. By zeta-potential measurements, the surface potential of these complexes was 25 mV at pH 5 and 0 mV at pH 7.4. In a tissue distribution study, the accumulation of NCL liposome/DNA complexes demonstrated around 10-fold higher in the liver as compared to the lung after intravenous injection. After treatment with the new complex, the mean plasma level of Factor VIII was 4.7 (⫾2.8) ng/mL. Only 0.86 (⫾0.3) ng/mL of Factor VIII could be detected after treatment with complexes without PEG and ligand surface modifications. No Factor VIII expression was detected with the conventional DDAB cationic liposome/DNA complexes under the same conditions. Administration of 100 g of naked plasmid DNA by 1-mL bolus injection volume and 0.2-mL small injection volume achieved plasma concentrations of Factor VIII of 1.2 (⫾0.3) ng/mL and 0.80 (⫾1.3) ng/mL, respectively. The results of this study indicate that the use of the NCL liposome/DNA complexes with specific surface modifications improves delivery to the desired tissues, facilitates internalization into target cells, and provides a useful approach for the systemic delivery of clinically relevant genes. Acknowledgements: The human Factor VIII plasmid and Factor VIII coatest assay were provided by Dr. Phil Scuderi’s group, Bayer Corporation.
27. Targeting of Lipid-Protamine-DNA (LPD) complexes using DSPE-PEG5000-LHRH or DSPEPEG5000-RGD Pierrot Harvie*, Ben Dutzar*, Todd Galbraith*, Sally Cudmore†, Dan O’Mahony†, Pervin Ankleseria*, Ralph Paul* *Targeted Genetics Corporation, 1100 Olive Way Seattle, WA 98101 USA †Elan Pharmaceutical Technologies, Dublin, Ireland Lipid-Protamine-DNA (LPD) lipopolyplexes have successfully been used for systemic gene transfer (Li et al. (1998) Gene Therapy, 5: 930-937). The use of polyethylene glycol (PEG)-modified lipids is well established for liposome encapsulated drugs and their ability to enhance the delivery of anti-cancer drugs to tumor sites has been proven (Lasic, (1998) Trends in Biotechnology 16; 307-32). Pegylated lipids are also known to control surface properties of cationic lipid-based gene transfer systems. However, pegylated lipid incorporation into lipid-DNA complexes causes a concentration dependent reduction of in vitro transfection activity, a result that can be partially attributed to a reduction in particle binding to cells (Harvie et al. J. Phar Sci. (2000) 89;5 652-663). In order to restore particle binding MOLECULAR THERAPY Vol. 3, No. 5, May 2001, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
IN
NON-VIRAL GENE TRANSFER
and target LPD to tumor cell, the lipid-peptide conjugates DSPE-PEG5k-LHRH (Leutinizing Hormone Release Hormone) or DSPE-PEG5k-RGD were generated and incorporated into a lipid-protamine-DNA (LPD) formulation. Addition of such lipidconjugated-ligands are compatible with LPD formation. Lipidconjugated-ligand incorporation into LPDs results in a dosedependent enhancement of LPD formulation binding to cells as well as a corresponding increase in luciferase expression following in vitro transfection of MDA-MB-231 and SKOV-3 cells. Maximum luciferase expression was observed at 10 mol percent lipidconjugated-ligand incorporation into the LPD formulations. Addition of 10 mol percent of DSPE-PEG5k-LHRH to an LPD formulation enhances transfection activity in vitro in MDA-MB231 cells by 285 fold over the base pegylated LPD formulation. DSPE-PEG5k-RGD incorporation into an LPD resulted in a transfection activity enhancement in vitro in MDA-MB-231 cells up to 100 fold. Moreover, transfection activity of LPD bearing DSPEPEG5k-LHRH can be abolished in a competition assay using 1000 fold excess of free LHRH. DSPE-PEG5k-RGD mediated transfection enhancement can also be abolished in a competition assay using 1000 fold excess of free RGD peptide, while an RGE control peptide had no effect. These data suggest that this those new systemic gene delivery formulations are extremely versatile for controlling LPD behavior and will lead to more effective cell specific targeting. This work was supported by Emerald Gene systems
28. Lipid/mu/DNA Complexes as Gene Transfer Agents M. Manvell*, T. Tagawa†, E. Perouzel†, A. Miller†, EWFW Alton* *Deptartment of Gene Therapy, Imperial College School of Medicine at the National Heart and Lung Institute, London, England †Imperial College Genetic Therapies Centre, Department of Chemistry, Imperial College, London, England For in vivo systemic gene transfer, both the volume in which DNA:lipid complexes are delivered and the potentially toxic effects of either component limit gene expression. We have assessed whether addition of the adenoviral mu protein to cationic lipids allows for more efficient gene transfer. Lipid/mu/DNA complexes (LMDs) were used to transfect COS 7 cells, with the ratio of Lipid:mu:DNA remaining constant at 12:0.6:1, whilst DNA and hence mu and lipid doses were varied. At the highest dose of DNA (5 g), standard DNA-DC-Cholesterol:DOPE (DCChol) lipid complexes (at 2:1 lipid:DNA) produced significantly (p⬍0.05, n⫽14) greater expression than equivalent doses of LMDs. However, at the lower dose of 0.5 g DNA the LMD complex performed significantly (p⬍0.01, n⫽8) better than DCChol with values equal to the transfection level seen with DCChol at 5 g DNA. When a time course was performed, LMD showed significant gene expression (assayed 48 hours later) after only 30 minutes incubation with the transfection mixture, whereas the DC-Chol complex only showed significant expression after 24 hours incubation. In the presence of 10% serum transfection efficiency was significantly (p⬍0.01, n⫽10) improved using LMDs (5 g DNA). In contrast, DC-Chol-mediated transfection was markedly reduced (p⬍0.01, n⫽10). 100% serum (5 g DNA) significantly (p⬍0.05, n⫽6) reduced LMD transfections compared to no serum but gene transfer could still be clearly seen. In contrast DC-Chol in the presence of 100% serum (at 2:1 lipid:DNA dose) showed no evidence of gene transfer. However, the 12:1 DC-Chol/DOPE:DNA control complexes performed as well as LMD in 100% serum, suggesting that the excess lipid in the LMD complex is responsible for the protective effect in serum. In conclusion, in vitro LMDs produced the same transfection efficiency as DC-Chol/DOPE complexes with one log order less DNA, and showed transfection at a much earlier time point. LMD could also produce gene transfer in the presence of 100% serum, due to the presence of excess lipid. These properties may make LMDs suitable for systemic delivery applications.
S11