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Publications (19)86.03 Total impact

  • Molecular Genetics and Metabolism 02/2013; 108(2):S20. · 2.83 Impact Factor
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    ABSTRACT: Niemann Pick type C (NPC) disease is a progressive neurodegenerative disease caused by mutations in NPC1 or NPC2, the gene products of which are involved in cholesterol transport in late endosomes. NPC is characterized by an accumulation of cholesterol, sphingomyelin and glycosphingolipids in the visceral organs, primarily the liver and spleen. In the brain, there is a redistribution of unesterified cholesterol and a concomitant accumulation of glycosphingolipids. It has been suggested that reducing the aberrant lysosomal storage of glycosphingolipids in the brain by a substrate reduction therapy (SRT) approach may prove beneficial. Inhibiting glucosylceramide synthase (GCS) using the iminosugar-based inhibitor miglustat (NB-DNJ) has been reported to increase the survival of NPC mice. Here, we tested the effects of Genz-529468, a more potent iminosugar-based inhibitor of GCS, in the NPC mouse. Oral administration of Genz-529468 or NB-DNJ to NPC mice improved their motor function, reduced CNS inflammation, and increased their longevity. However, Genz-529468 offered a wider therapeutic window and better therapeutic index than NB-DNJ. Analysis of the glycolipids in the CNS of the iminosugar-treated NPC mouse revealed that the glucosylceramide (GL1) but not the ganglioside levels were highly elevated. This increase in GL1 was likely caused by the off-target inhibition of the murine non-lysosomal glucosylceramidase, Gba2. Hence, the basis for the observed effects of these inhibitors in NPC mice might be related to their inhibition of Gba2 or another unintended target rather than a result of substrate reduction.
    Molecular Genetics and Metabolism 02/2012; 105(4):621-8. · 2.83 Impact Factor
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    ABSTRACT: In mice, liver-restricted expression of lysosomal enzymes from adeno-associated viral serotype 8 (AAV8) vectors results in reduced antibodies to the expressed proteins. To ask whether this result might translate to patients, nonhuman primates (NHPs) were injected systemically with AAV8 encoding α-galactosidase A (α-gal). As in mice, sustained expression in monkeys attenuated antibody responses to α-gal. However, this effect was not robust, and sustained α-gal levels were 1-2 logs lower than those achieved in male mice at the same vector dose. Because our mouse studies had shown that antibody levels were directly related to expression levels, several strategies were evaluated to increase expression in monkeys. Unlike mice, expression in monkeys did not respond to androgens. Local delivery to the liver, immune suppression, a self-complementary vector and pharmacologic approaches similarly failed to increase expression. While equivalent vector copies reached mouse and primate liver and there were no apparent differences in vector form, methylation or deamination, transgene expression was limited at the mRNA level in monkeys. These results suggest that compared to mice, transcription from an AAV8 vector in monkeys can be significantly reduced. They also suggest some current limits on achieving clinically useful antibody reduction and therapeutic benefit for lysosomal storage diseases using a systemic AAV8-based approach.
    Molecular Therapy 06/2011; 19(11):1999-2011. · 7.04 Impact Factor
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    ABSTRACT: The neuropathic glycosphingolipidoses are a subgroup of lysosomal storage disorders for which there are no effective therapies. A potential approach is substrate reduction therapy using inhibitors of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide and related glycosphingolipids that accumulate in the lysosomes. Genz-529468, a blood-brain barrier-permeant iminosugar-based GCS inhibitor, was used to evaluate this concept in a mouse model of Sandhoff disease, which accumulates the glycosphingolipid GM2 in the visceral organs and CNS. As expected, oral administration of the drug inhibited hepatic GM2 accumulation. Paradoxically, in the brain, treatment resulted in a slight increase in GM2 levels and a 20-fold increase in glucosylceramide levels. The increase in brain glucosylceramide levels might be due to concurrent inhibition of the non-lysosomal glucosylceramidase, Gba2. Similar results were observed with NB-DNJ, another iminosugar-based GCS inhibitor. Despite these unanticipated increases in glycosphingolipids in the CNS, treatment nevertheless delayed the loss of motor function and coordination and extended the lifespan of the Sandhoff mice. These results suggest that the CNS benefits observed in the Sandhoff mice might not necessarily be due to substrate reduction therapy but rather to off-target effects.
    PLoS ONE 01/2011; 6(6):e21758. · 3.53 Impact Factor
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    ABSTRACT: Liver-directed gene therapy with adeno-associated virus (AAV) vectors effectively treats mouse models of lysosomal storage diseases (LSDs). We asked whether these results were likely to translate to patients. To understand to what extent preexisting anti-AAV8 antibodies could impede AAV8-mediated liver transduction in primates, commonly preexposed to AAV, we quantified the effects of preexisting antibodies on liver transduction and subsequent transgene expression in mouse and nonhuman primate (NHP) models. Using the highest viral dose previously reported in a clinical trial, passive transfer of NHP sera containing relatively low anti-AAV8 titers into mice blocked liver transduction, which could be partially overcome by increasing vector dose tenfold. Based on this and a survey of anti-AAV8 titers in 112 humans, we predict that high-dose systemic gene therapy would successfully transduce liver in >50% of human patients. However, although high-dose AAV8 administration to mice and monkeys with equivalent anti-AAV8 titers led to comparable liver vector copy numbers, the resulting transgene expression in primates was ~1.5-logs lower than mice. This suggests vector fate differs in these species and that strategies focused solely on overcoming preexisting vector-specific antibodies may be insufficient to achieve clinically meaningful expression levels of LSD genes using a liver-directed gene therapy approach in patients.
    Molecular Therapy 11/2010; 18(11):1983-94. · 7.04 Impact Factor
  • Molecular Genetics and Metabolism - MOL GENET METAB. 01/2010; 99(2).
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    ABSTRACT: In an effort to improve the efficiency of cationic lipid mediated gene transfer, over 90 novel cationic lipids of diverse structural types were synthesized and evaluated in vitro. Four cationic lipids derived from phospholipids were examined. The most promising cationic lipid formulations were tested in vivo by intranasal or transtracheal instillation into the lungs of BALB/c mice. The most active formulations gave CAT reporter gene expression levels which are greater than 500 fold over that which could be attained using free DNA alone. Certain cationic lipid formulations have been shown to facilitate substantial expression of the CFTR (cystic fibrosis transmembrane conductance regulator) gene in vitro as determined by the SPQ and Ussing Chamber assays.
    Phosphorus Sulfur and Silicon and the Related Elements 08/2008; Sulfur(and Silicon and the Related Elements):289-292. · 0.60 Impact Factor
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    ABSTRACT: Gaucher disease is the most common of the lysosomal storage disorders. The primary manifestation is the accumulation of glucosylceramide (GL-1) in the macrophages of liver and spleen (Gaucher cells), due to a deficiency in the lysosomal hydrolase glucocerebrosidase (GC). A Gaucher mouse model (D409V/null) exhibiting reduced GC activity and accumulation of GL-1 was used to evaluate adeno-associated viral (AAV)-mediated gene therapy. A recombinant AAV8 serotype vector bearing human GC (hGC) was administered intravenously to the mice. The levels of hGC in blood and tissues were determined, as were the effects of gene transfer on the levels of GL-1. Histopathological evaluation was performed on liver, spleen and lungs. Vector administration to pre-symptomatic Gaucher mice resulted in sustained hepatic secretion of hGC at levels that prevented GL-1 accumulation and the appearance of Gaucher cells in the liver, spleen and lungs. AAV administration to older mice with established disease resulted in normalization of GL-1 levels in the spleen and liver and partially reduced that in the lung. Analysis of the bronchoalveolar lavage fluid (BALF) from treated mice showed significant correction of the abnormal cellularity and cell differentials. No antibodies to the expressed hGC were detected following a challenge with recombinant enzyme suggesting the animals were tolerized to human enzyme. These data demonstrate the effectiveness of AAV-mediated gene therapy at preventing and correcting the biochemical and pathological abnormalities in a Gaucher mouse model, and thus support the continued consideration of this vector as an alternative approach to treating Gaucher disease.
    The Journal of Gene Medicine 07/2006; 8(6):719-29. · 2.16 Impact Factor
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    ABSTRACT: In murine models of lysosomal storage diseases, sustained hepatocyte-restricted expression and secretion of the therapeutic enzyme is capable of correcting the peripheral pathology associated with the storage product. For example, systemic delivery of a recombinant AAV8 vector encoding human alpha-galactosidase A to Fabry mice resulted in complete clearance of the glycosphingolipid storage product in peripherally affected tissues when sustained serum alpha-galactosidase A levels were in excess of 1 ug/ml. In humans, transient blood levels of human FIX have been obtained after hepatocyte transduction with an AAV2 vector. The abbreviatedexpression was attributed to cytotoxic lymphocyte (CTL) recognition of viral capsid epitopes on the transduced cells. In view of these potential immune-based limitations on expression, we have asked whether we could generate prolonged circulating levels of human alpha-galactosidase A following delivery of an AAV2/8 vector encoding human alpha-galactosidase A under control of a hepatocyte-restricted promoter to immune suppressed rhesus macaques. We have compared systemic delivery using a peripheral vein approach and local delivery using a hepatic vein approach that was developed in a rabbit model. The hepatic vein approach uses a balloon catheter to isolate a lobe of the liver and block blood flow into the vena cava. We then flush the vasculature of the lobe retrograde to blood flow to reduce the concentration of any resident anti-viral antibodies and to allow the viral vector to |[ldquo]|dwell|[rdquo]| within the lobe for a defined period after delivery. A three month immune suppression regime was used to blunt any neutralizing immune responses to virally-transduced cells or human alpha-galactosidase A. After removing immune suppression, animals will be challenged with purified human alpha- galactosidase A to assess their degree of tolerance to this foreign protein. Although this experiment is not yet complete, we will present up-to-date expression and toxicity data comparing the two delivery techniques.
    Molecular Therapy 05/2006; · 7.04 Impact Factor
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    ABSTRACT: Gaucher disease is the most common of the >40 described lysosomal storage disorders. The primary manifestation of Gaucher disease is the accumulation of glucosylceramide (GL-1), predominantly in the macrophages of liver and spleen, due to a genetic defect in the lysosomal hydrolase glucocerebrosidase (GC). A mouse model of Type I Gaucher disease (D409V/null) was used to assess the efficacy of AAV8-mediated gene therapy. This mouse model exhibits only ∼5% of normal GC activity in visceral tissues resulting in elevated GL-1 levels and the appearance of enlarged macrophages (Gaucher cells) in liver, lung, and spleen at 3 months of age. The AAV vector (AAV8/DC172-hGC) used for these studies contained a hepatocyte-restricted promoter, DC172 (human α1-microglobulin enhancer, human α1-anti-trypsin promoter), to drive the expression of human GC. Intravenous administration of 3×1011 drp of AAV8/DC172-hGC into 4-week old D409V/null mice (prior to development of disease pathology) generated high levels of GC in the liver and serum, and subsequent uptake by the spleen and lung. Expression was sustained for the duration of the study (6 months) and was effective at preventing both the accumulation of GL-1 in the tissues and the consequent onset of Gaucher disease symptoms. A subsequent study in older mice (4-month old) with established disease pathology showed that administration of a similar dose of AAV8/DC172-hGC was also effective at reversing GL-1 storage in the affected tissues. Furthermore, AAV-treated mice were largely devoid of the lipid-engorged macrophages that were frequently observed in vehicle-treated D409V/null mice. A dose-response study, in which 5-month old mice received 3×1011, 1×1011 or 3×1010 drp of AAV8/DC172-hGC, yielded high and sustained expression of GC in all treatment groups. Three months post-injection, both the 3×1011 and 1×1011 drp dosed mice demonstrated a dramatic reduction in GL-1 levels and the number of Gaucher storage cells in liver, lung and spleen. Treatment also corrected the abnormal pathology, cell differentials and MIP-1α levels in the lungs of the D409V/null mice in a dose-dependent manner. Additionally, no antibodies to GC were detected, in accordance with previous AAV gene therapy studies using hepatocyte-restricted promoters. These data demonstrate the effectiveness of AAV-mediated gene therapy at preventing and correcting the biochemical and pathological abnormalities in the mouse model and thus support its continued development in the treatment of Gaucher Disease.Employee and shareholder of Genzyme Corporation
    Molecular Therapy 01/2005; · 7.04 Impact Factor
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    ABSTRACT: Molecular Therapy (2004) 9, S324–S325; doi: 10.1016/j.ymthe.2004.06.762 854. Feasibility of AAV-Mediated Gene Therapy Examined Using a New Murine Model (D409V/null) of Gaucher Disease John Marshall1, Kerry McEachern1, Jennifer B. Nietupski1, Julie A. Cavanagh Kyros1, Donna Armentano1, Gregory A. Grabowski2 and Seng H. Cheng11Genzyme Corporation, Framingham, MA2Divisions of Human Genetics and Pathology, Cincinnati Children's Hospital Research Foundation, Cincinnati, OH
    Molecular Therapy 04/2004; · 7.04 Impact Factor
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    ABSTRACT: Progress towards developing gene therapy for Gaucher disease has been hindered by the lack of an animal model. Here we describe a mouse model of Gaucher disease which has a chemically induced deficiency of glucocerebrosidase and that accumulates elevated levels of glucosylceramide (GL-1) in the lysosomes of Kupffer cells. Administration of mannose-terminated glucocerebrosidase (Cerezyme) resulted in the reduction of GL-1 levels in the livers of these animals. Gene transduction of hepatocytes with a plasmid DNA vector encoding human glucocerebrosidase (pGZB-GC) generated high-level expression and secretion of the enzyme into systemic circulation with consequent normalization of Kupffer cell GL-1 levels. This suggested that the de novo synthesized and unmodified enzyme produced by hepatocyte transduction was also capable of being delivered to the cells that are primarily affected in Gaucher disease. Immunolocalization studies also revealed that preferential transduction and expression of human glucocerebrosidase in the Kupffer cells with subsequent reduction in the GL-1 levels could be attained with a low dose of a recombinant adenoviral vector encoding the human enzyme (Ad2/CMV-GC). This observation raises the possibility of gene therapy for Gaucher disease that involves directly transducing the affected histiocytes using recombinant adenoviral vectors. Together, these data demonstrate the potential for use of in vivo gene therapy vectors for treating Gaucher disease.
    Molecular Therapy 09/2002; 6(2):179-89. · 7.04 Impact Factor
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    ABSTRACT: A major limitation associated with systemic administration of cationic lipid:plasmid DNA (pDNA) complexes is the vector toxicity at the doses necessary to produce therapeutically relevant levels of transgene expression. Systematic evaluation of these toxicities has revealed that mice injected intravenously with cationic lipid:pDNA complexes develop significant, dose-dependent hematologic and serologic changes typified by profound leukopenia, thrombocytopenia, and elevated levels of serum transaminases indicative of hepatocellular necrosis. Vector administration also induced a potent inflammatory response characterized by complement activation and the induction of the cytokines IFN-gamma, TNF-alpha, IL-6, and IL-12. These toxicities were found to be transient, resolving with different kinetics to pretreatment levels by 14 days posttreatment. The toxic syndrome observed was independent of the cationic lipid:pDNA ratio, the cationic lipid species, and the level of transgene expression attained. Mechanistic studies determined that neither the complement cascade nor TNF-alpha were key mediators in the development of these characteristic toxicities. Administration of equivalent doses of the individual vector components revealed that cationic liposomes or pDNA alone did not generate the toxic responses observed with cationic lipid:pDNA complexes. Only moderate leukopenia was associated with administration of cationic liposomes or pDNA alone, while only mild thrombocytopenia was noted in pDNA-treated animals. These results establish a panel of objective parameters that can be used to quantify the acute toxicities resulting from systemic administration of cationic lipid:pDNA complexes, which in turn provides a means to compare the therapeutic indices of these vectors.
    Human Gene Therapy 01/2001; 11(18):2493-513. · 4.02 Impact Factor
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    ABSTRACT: Enhanced gene transduction to the lung using cationic lipids could be attained through optimization of the structure of the lipids and the formulation of the cationic lipid:plasmid DNA (pDNA) complexes. We have expanded on our earlier observation of the importance of the structural orientation of the cationic lipid headgroup. Through the synthesis of a number of matched pairs of cationic lipids differing only in the configuration of their headgroup, we confirmed that those harboring a T-shape headgroup are more active than their linear counterparts, at least when tested in the lungs of BALB/c mice. Additionally, we demonstrated that not only are the structural considerations of these cationic lipids important, but also their protonation state, the free base being invariably more active than its salt counterpart. The salt forms of cationic lipids bound pDNA with greater avidity, which may have affected their subsequent intracellular dissolution and transit of the pDNA to the nucleus. Inclusion of a number of frequently used solutes in the vehicle severely inhibited the gene transfection activity of the cationic lipids. The selection of neutral co-lipids was also an important factor for overall transfection activity of the formulation, with significant gains in transfection activity realized when diphytanoylphosphatidylethanolamine or dilinoleoylphosphatidylethanolamine were used in lieu of dioleoylphosphatidylethanolamine. Finally, we showed that a transacylation reaction could occur between the cationic lipid and neutral co-lipid which reduced the transfection activity of the complexes. It is the hope that as our understanding of the many factors that influence the activity of these cationic lipid:pDNA complexes improves, formulations with much greater potency can be realized for use in the treatment of pulmonary diseases.
    Journal of Drug Targeting 02/2000; 7(6):453-69. · 2.77 Impact Factor
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    ABSTRACT: In human airways, the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is predominantly expressed in serous cells of the tracheobronchial glands. Despite considerable evidence that submucosal glands are important contributors to the pathophysiology of CF lung disease, most attempts at CFTR gene transfer have primarily targeted airway surface epithelial cells. In this study, we systematically evaluated CFTR gene transfer into cultures of immortalized CF human tracheobronchial submucosal gland (6CFSMEO) cells using adenovirus and cationic lipid vectors. We found that the efficiency of adenovirus-mediated gene transfer was comparable in 6CFSMEO and CFT1 cells (a surface airway epithelial cell line isolated from a subject with CF). So was the ranking order of adenovirus vectors containing different enhancers/promoters (CMV > E1a approximately phosphoglycerokinase), as determined by both X-Gal staining and quantitative measurement of beta-galactosidase activity. Further, we provide the first demonstration that cationic lipids mediate efficient gene transfer into 6CFSMEO cells in vitro. The transfection efficiency at optimal conditions was higher in 6CFSMEO than in CFT1 cells. Finally, either infection with adenoviral vectors or transfection with cationic lipid:plasmid DNA complexes encoding CFTR significantly increased chloride (Cl-) permeability, as assessed using the 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ) fluorescence assay, indicating restoration of functional CFTR Cl- channel activity. These data show that although the mechanisms of transfection may be different between the two cell types, 6CFSMEO cells are as susceptible as CFT1 cells to transfection by adenoviral and cationic-lipid gene transfer vectors.
    American Journal of Respiratory Cell and Molecular Biology 07/1999; 20(6):1107-15. · 4.15 Impact Factor
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    ABSTRACT: Gene therapy is a promising strategy to modify ischemia-reperfusion injury and rejection after transplantation. We evaluated variables that may affect ex vivo gene transfer to rat lung isografts. Left lungs were harvested and perfused via the pulmonary vein with chloramphenicol acetyltransferase complementary deoxyribonucleic acid complexed with cationic liposomes. Several variables were examined: (1) Influence of temperature: In group I (n = 4), grafts were stored for 4 hours at 23 degrees C and transplanted. Chloramphenicol acetyltransferase activity was assessed on postoperative day 2. In groups II and III (n = 4), grafts were stored at 10 degrees and 4 degrees C, respectively. Arterial oxygen tension and inflammatory infiltrate were also determined. (2) Influence of storage time: Grafts were preserved at 10 degrees C for 1, 2, 3, 4 (n = 4), and 10 hours (n = 5). chloramphenicol acetyltransferase activity was assessed on postoperative day 2. (3) Rapidity and duration of transgene expression: Grafts were preserved at 10 degrees C for 1 hour and then transplanted. Chloramphenicol acetyltransferase activity was assessed 2, 4, 6, 12, and 24 hours and 2, 7, 14, 21, and 28 days after implantation. Chloramphenicol acetyltransferase expression was apparently less in lungs transfected at 4 degrees C than in those transfected at 10 degrees and 23 degrees C. Storage for 1 hour at 10 degrees C was sufficient to yield significant expression. Increasing the exposure time to 10 hours did not increase toxicity. There were no differences in arterial oxygen tension between transfected and nontransfected lungs. Chloramphenicol acetyltransferase expression was detected for at least 28 days. Ex vivo liposome-mediated transfection of lung isografts can be achieved after a short time of cold storage, with minimal toxicity.
    Journal of Thoracic and Cardiovascular Surgery 02/1998; 115(1):38-44. · 3.53 Impact Factor
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    ABSTRACT: Our objective were to determine the feasibility, efficacy, and safety of in vivo and ex vivo liposome-mediated gene transfer to lung isografts. Fischer rats were divided into three main groups: (1) Nontransplant setting: Liposome-chloramphenicol acetyl transferase cDNA was intravenously injected, and lungs were harvested at different time points: 2, 6, 12, and 24 hours; 2, 5, 8, and 21 days (n = 3). Chloramphenicol acetyl transferase activity was determined in lungs, hearts, livers, and kidneys. The distribution and type of transfected cells were evaluated by in situ hybridization. Lung toxicity was assessed by arterial oxygen tension, histology, and tumor necrosis factor-alpha levels. (2) In vivo graft transfection: Left lungs were transplanted 6 hours, 4 hours, and 15 minutes after intravenous injection and were assessed for chloramphenicol acetyl transferase activity and arterial oxygen tension on postoperative day 2. (3) Ex vivo graft transfection: Grafts were infused ex vivo with either 660 micrograms (n = 3) or 330 micrograms (n = 3) of DNA complexed to liposomes and stored at 10 degrees C for 4 hours. Chloramphenicol acetyl transferase activity was assessed 44 hours after transplantation. Transgene expression was detected in endothelial cells, macrophages, and interstitial cells. Chloramphenicol acetyl transferase activity was present as early as 2 hours, increased significantly between 6 hours and 8 days, and then decreased to minimal levels by 21 days. Chloramphenicol acetyl transferase activity was greatest in donor lungs and hearts and minimal in livers and kidneys. Arterial oxygen tension was normal in treated animals. Inflammation was minimal, and tumor necrosis factor-alpha levels increased only sevenfold in treated animals. In vivo and ex vivo liposome-mediated gene transfer to lung isografts allows significant transgene expression with minimal effects on graft function.
    Journal of Thoracic and Cardiovascular Surgery 12/1997; 114(5):783-91; discussion 791-2. · 3.53 Impact Factor
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    ABSTRACT: Effective gene therapy for lung tissue requires the use of efficient vehicles to deliver the gene of interest into lung cells. When plasmid DNA encoding chloramphenicol acetyltransferase (CAT) was administered intranasally to BALB/c mice without carrier lipids, CAT activity was detected in mouse lung extracts. Plasmid DNA delivered with optimally formulated commercially available transfection reagents expressed up to 10-fold more CAT activity in lung than observed with naked DNA alone. Liposome formulations consisting of (+/-)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis (dodecyloxy)-1-propanaminium bromide (GAP-DLRIE) plus the neutral colipid dioleoylphosphatidylethanolamine (DOPE) enhanced CAT expression by more than 100-fold relative to plasmid DNA alone. A single administration of GAP-DLRIE liposome-CAT DNA complexes to mouse lung elicited peak expression at days 1-4 posttransfection, followed by a gradual return to baseline by day 21 postadministration. Readministration of GAP-DLRIE liposome CAT complexes at day 21 led to another transient peak of reporter gene expression. Histological examination of lungs treated with GAP-DLRIE complexed beta-galactosidase DNA revealed that alveolar epithelial cells were the primary locus of expression and that up to 1% of all alveoli contained epithelial cells expressing the transgene.
    Proceedings of the National Academy of Sciences 11/1996; 93(21):11454-9. · 9.81 Impact Factor
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    ABSTRACT: Cationic lipid-mediated gene transfer of cystic fibrosis transmembrane conductance regulator (CFTR) cDNA represents a promising approach for treatment of cystic fibrosis (CF). Here, we report on the structures of several novel cationic lipids that are effective for gene delivery to the lungs of mice. An amphiphile (#67) consisting of a cholesterol anchor linked to a spermine headgroup in a "T-shape" configuration was shown to be particularly efficacious. An optimized formulation of #67 and plasmid vector encoding chloramphenicol acetyl-transferase (CAT) was capable of generating up to 1 microgram of CAT enzyme/lung following intranasal instillation into BALB/c mice. This represents a 1,000-fold increase in expression above that obtained in animals instilled with naked pDNA alone and is greater than 100-fold more active than cationic lipids used previously for CFTR gene expression. When directly compared with adenovirus-based vectors containing similar transcription units, the number of molecules of gene product expressed using lipid-mediated transfer was equivalent to vector administration at multiplicities of infection ranging from 1 to 20. The level of transgene expression in the lungs of BALB/c mice peaked between days 1 and 4 post-instillation, followed by a rapid decline to approximately 20% of the maximal value by day 7. Undiminished levels of transgene expression in the lung could be obtained following repeated intranasal administration of #67:DOPE:pCF1-CAT in nude mice. Transfection of cells with formulations of #67:DOPE:pCF1-CFTR generated cAMP-stimulated CFTR chloride channel and fluid transport activities, two well-characterized defects associated with CF cells. Taken together, the data demonstrate that cationic lipid-mediated gene delivery and expression of CFTR in CF lungs is a viable and promising approach for treatment of the disease.
    Human Gene Therapy 10/1996; 7(14):1701-17. · 4.02 Impact Factor