538 cytoskeletal involvement in the albumin facilitated lipofection gene delivery strategy

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Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts

LIPID MEDIATED GENE TRANSFER

Cationic lipid:Protamine:DNA (LPD) formulations and at a specific

ratio of >= 3 mg PPAA/mg DNA, an optimal enhancement of in

vitro transfection activity was observed Formulations containing

PPAA demonstrated a shift in their zeta potential from a negative

zeta potential at pH 7.4 to a positive zeta potential at pH 4.2

indicating that the predicted charge shift associated with the polymer

at low pH was operational in the formulation context Incorporation

of PPAA into LPD formulations enhanced the in vitro transfection

of KB cells by 1-2 logs over the unmodified formulation However,

incorporation of PPAA in these formulations increased the mean

formulation particle diameter from ~200 nm up to 800 nm This

size alteration was prevented when the LPD formulations were

modified through the incorporation of DSPE-PEG5K (LPD-PEG)

or DSPE-PEG5K-Folate (LPD-PEG-Folate) PPAA addition to

LPD-PEG formulations enhanced transfection potency 2 logs over

unmodified LPD-PEG formulations More interestingly, a 3 log

transfection enhancement was observed for LPD-PEG-Folate

formulations containing PPAA, indicating a synergistic effect

between the polymer and the Folate ligand These transfection

enhancements were maintained or improved upon even after

incubation of these formulations for 1h incubation at 37ºC in mouse

serum prior to transfection This suggests that these new formulations

may be viable for in vivo application Addition of Chloroquine to

transfections inhibited the observed PPAA mediated transfection

enhancement This supported the possible importance of the

endosomal acidification in PPAA mediated transfection

enhancement Cumulatively, this data suggests that this modification

of a multifunctional gene delivery system represents an additional

step in the generation of synthetic delivery systems with greater

transfection efficiencies

Acute Inflammatory Response to Nonviral Vectors

Hongmei Zhao,1 I.-Huan Wu,1 Hiraoki Hemmi,2 Shizuo Akira,2

Seng H Cheng,1 Ronald K Scheule,1 Nelson S Yew.1

1 Gene Transfer Research, Genzyme Corporation, Framingham,

MA; 2 Department of Host Defense, Research Institute for

Microbial Diseases, Osaka University, Suita, Osaka, Japan.

We have shown previously that cationic lipid-plasmid DNA

(pDNA) complexes containing pDNA vectors that have been largely

depleted of CpG motifs have reduced toxicity when delivered

systemically However, several CpGs remain in these vectors and

the acute toxicity is not negligible, especially at higher doses of

complex To determine which toxicities can or cannot be resolved by

completely eliminating CpG-mediated stimulation, we measured

the response to systemically delivered complexes in transgenic mice

that are deficient in the Toll-like 9 (TLR9) receptor, which is the

receptor that recognizes immunostimulatory CpG motifs One day

after injecting complex, the proinflammatory cytokines IL-12, IL-6,

and IFN-gamma remained at near background levels in the

TLR9-/-mice A significant decrease in adverse hematological changes and

reduced elevations of the liver enzymes ALT and AST were also

observed in the TLR9-/- mice compare to normal mice, with a

concomitant marked improvement in survival However, a

pronounced loss of lymphocytes and platelets was still observed in

the TLR9-/- mice at higher doses, similar to that seen in normal

C57BL/6 mice We also measured the toxicity in normal mice of

systemically delivered complexes containing non-CpG

oligonucleotides or DNA fragments that are nearly devoid of CpGs

Although ALT and AST levels were reduced, a loss of lymphocytes

and platelets was observed akin to that seen in the TLR9-/- mice

Taken together, these results suggest that signaling through the TLR9

receptor contributes to some but not all of the toxic responses

associated with systemic delivery of cationic lipid-pDNA complexes

The results also imply that a completely non-CpG pDNA vector

will have limited but nevertheless beneficial effects on decreasing liver damage and increasing the safety of systemically delivered nonviral vectors

Transgene Expression in A549 Cells through the Activation of the Src and ERK Kinase Pathway

Rajesh R Nair,1 Lindsay A Schwarz.1

1 Dept of PPS, University of Houston, Houston, TX, United States.

Colchicine, a microtubule-disrupting agent, has been proposed to enhance transgene expression in cells by inhibiting the endo-lysosomal fusion We found that in several different human cell lines, including A549 human lung adenocarcinoma cells, 1 hr treatment with 5 μM colchicine resulted in a 15-30-fold increase in transgene expression Increased transgene expression was evident even in cells not treated until 12 hrs after transfection, with 5 μM colchicine In addition, when cells were pulsed for 1 hr with 5 μM colchicine and transfected after 18 hrs post-treatment, enhanced transgene expression was still observed These data suggested that the colchicine-mediated increase

in transgene expression occurs through induction of a facilitating factor and not through inhibition of endo-lysosomal fusion To test whether microtubular depolymerization was critical to the activation

of this factor, cells were pretreated with a microtubular stabilizer, paclitaxel, 3 hrs before colchicine Paclitaxel pretreatment dose-dependently and significantly inhibited the colchicine-induced increase in transgene expression, suggesting that microtubular disruption is pivotal for the activation of the factor facilitating the increase in transgene expression Colchicine also activates the src family kinases which, in turn, leads to the sustained activation of ERK1/2 To investigate the significance of this pathway in the colchicine-enhanced transgene expression, we treated cells with PP1,

a specific src kinase inhibitor PP1 dose dependently and significantly inhibited the colchicine-induced increase in transgene expression, suggesting a role of src kinase in this pathway Further, PD98059 an inhibitor of ERK1/2, also dose dependently and significantly decreased colchicine-enhanced transgene expression The ability of PD98059 to inhibit ERK1/2 was confirmed by probing for activated ERK1/2 by Western blotting Since ERK1/2 regulate transcription

of certain cellular genes, we tested if new transcription is needed for the colchicine-enhanced transgene expression Actinomycin D, an inhibitor or transcription, dose-dependently and significantly inhibited the colchicine effect In summary, colchicine appears to increase transgene expression via a signaling pathway initiated by microtubular depolymerization and mediated by activation of src and ERK kinases, which subsequently modulate transcription

Albumin-Facilitated Lipofection Gene Delivery Strategy

Robert W Arpke,1 Pi-Wan Cheng.1,2

1 Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE; 2 Eppley Institute for Cancer Research, University of Nebraska Medical Center, Omaha, NE.

Our lab has shown that supplementing liposome with a protein, such as transferrin, insulin, or lectins can circumvent the limitation

of low transfection efficiency of liposome-based gene delivery strategies Recently, we found that human serum albumin (HSA) can enhance the lipofection efficiency 10-20 fold in a human pancreatic cancer cell line, Panc 1 The purpose of this study is to characterize the albumin-facilitated lipofection gene delivery strategy and elucidate its gene targeting mechanism By agarose gel electrophoresis, the amount of DMRIE-Cholesterol (DC) required

to fully complex 1 μg of pCMVlacZ in the presence of 0.05 μg HSA was determined to be 5.5 μg Upon addition of increasing amounts

of HSA, DNA was excluded from the complexes, corresponding

Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts

Copyright © The American Society of Gene Therapy

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LIPID MEDIATED GENE TRANSFER

with a decrease in transfection efficiency Approximately 14% of

the [125I]HSA in the optimal formulation was in DC-HSA-DNA

ternary complexes as shown by Sepharose 4B column

chromatography Measured by light scattering, the size of the

transfection complexes in the optimal formulation was found to be

2-2.5 times that of DC as well as DC plus DNA complexes

Transmission electron microscopy examination of cells transfected

with a formulation prepared with [20 nm-gold]albumin, DC, and

[10 nm-gold]DNA showed DNA in an extended conformation at

the cell surface and co-localization of albumin and DNA

intracellularly The data suggest that albumin prevents DNA

condensation by the liposome, co-transports with the DNA, and

facilitates nuclear targeting of the DNA Despite its extended nature

in the transfection complexes, the DNA was resistant to DNase

treatment To examine the mechanism of endocytosis and intracellular

trafficking of the transfection complexes, various inhibitors were

utilized Pretreatment with chlorpromazine and excess HSA caused

a 30% and 25% decrease of transfection efficiency, respectively,

whereas no apparent effect was observed for filipin complex The

results suggest involvement of an albumin receptor and

clathrin-coated pits, but not caveolae in endocytosis of the transfection

complexes Treatment with the three classes of microtubule

modifying-agents, such as colchicine, paclitaxel, and vinblastine

enhanced the transfection efficiency 2-, 2-, and 1.5-fold, respectively

However, an actin inhibitor, cytochalasin B, caused 60% inhibition

We conclude that in our gene delivery strategy, albumin and DNA

are co-transported to the nucleus via an actin-dependent and

microtubule-independent mechanism Incorporation of cytoskeletal

modulating agents in the albumin-facilitated lipofection gene delivery

strategy should further the potential utilization of this non-viral

vector for in vivo gene therapy.

Gene Delivery in Three Dimensional Culture

Alicia L Bertone,1 Rhonda Schreiber,2 Stephen B Trippel.3

1 Veterinary Clinical Sciences, The Ohio State University,

Columbus, OH, United States; 2 Selective Genetics, Inc, San

Diego, CA, United States; 3 Orthopedics, University of Indiana,

Indianapolis, IN, United States.

Several morphogens have the ability to enhance cellular

proliferation (ie, FGF-2) and expression of proteoglycan [PG] and

type-2 collagen (ie, IGF-1 and BMP-2) in chondrocytes Delivery

of these genes can be therapeutic Nonviral (vs viral) gene delivery

offers advantages, but suffers from lower transfection Our study

(1) optimized direct DNA transfer in a 3-D cell system that can be

implanted in vivo using plasmid DNA and (2) evaluated hFGF,

hIGF1 and hBMP2 to promote chondrocyte proliferation and

expression of matrix using a 3-D collagen suspension Chondrocytes

from the knee joint of calves were suspended in constructs of known

cell density with the desired DNA and lipid [Lipofectamine2000]

concentration in type-1 collagen gel and cultured for 24 days

Optimal cell density [0.5, 1, and 2 x 106 cells/well], pDNA/well [0,

0.9, 1.86, 4.65, 9.3, 18.6, 37.2, and 74.4 ug DNA], and lipid reagent

[2:1, 3:1 and 6:1 DNA/Lipid ratio] for transfection were quantified

with plucerifase [pg/ml] and %GFP+ cells

%GFP+cells for each of 3 lipid/dna ratios

ugdna Lipo/dna ratio %pos Lipo/dna ratio %pos Lipo/dna ratio %pos

Based on the results of the optimization studies, 5 x 104 cells/

well, 37.2 ug DNA, and a 3:1 lipid/DNA ratio (111.6ug lipid) were

used in the following morphogen groups: (1) No DNA, No lipid

Control [NoL/NoD]; 2) No DNA, lipid Control [L/NoD]; 3)

pLuciferase, lipid; 5) phFGF2, lipid; 6) phIGF1,lipid; and 7)phBMP2, lipid Outcome measures included cell viability, tranfection efficiency (%), cell proliferation (doubling time), protein expression (pluciferase), proteoglycan production (Alcian blue stain intensity) and morphology (Eosin stain)

Gene transfer and expression occurred only when supplemented with lipid agent Growth factors promoted the health of chondrocytes

in this 3-D culture system hIGF and HBMP2 promoted proteoglycan production to the greatest extent and had the maximal morphology scores FGF and BMP2 promoted cell health and growth

to the greatest extent based on appearance, cell number and viability Plasmid delivery of morphogens enhanced cell proliferation, viability in the presence of lipid agent, and maintenance of chondrogenic phenotype Group Doubling (hrs) Day 7- % Viability Alcian stain Morph Score

Lipid agent at the concentrations necessary to support acceptable direct plasmid gene transfer to chondrocytes (~15%) is biocompatible with the cells in the shorter term (up to 4 days), but prolonged exposure (7 days) resulted in altered morphology and cell death Transfection efficiency in this 3-D cell suspension produced expression comparable to other lipid based methods Relevant effectiveness in vivo may be achieved in 3-D cellular constructs without vector delivery

Acknowledgement: CH Evans for consultation/facilities

Formulation of Lower-Charged, Highly Transfecting Cationic Liposomes for Gene Delivery

Steven Fletcher, Michael R Jorgensen, Kostas Kostarelos, Andrew D Miller

1 Imperial College Genetic Therapies Centre, Department of Chemistry, Imperial College London, London, United Kingdom.

Towards enhancing the in vivo gene transfer efficiency of cationic

liposomes, it is necessary to reduce the magnitude of the cationic charge of the liposome-based systems, without impairing transfection To this end, we have prepared a series of monoacetylenic analogues of the neutral, helper lipid dioleoyl-L- α-phosphatidylethanolamine (DOPE) in the belief that these structural changes should raise the Lα / HII phase transition temperature, Th, above that of DOPE (10 °C) Provided Th does not exceed the physiological temperature, then not only should the fusogenicity of the original DOPE molecule be maintained but the increased stability

at lower temperatures should enable the preparation of lower-charged, yet highly transfecting, cationic liposomes through enhanced DOPE-analogue content

Through31P NMR spectroscopy, we report the phase behaviour

of these analogues As predicted, all Lα / HII phase transition temperatures are greater than that of DOPE, but only one analogue, namely DO(9-yne)PE, adopts the HII phase below the physiological temperature (Th = 25 – 30 °C) The phase behaviour of this lipid, relative to DOPE, prompted us to attempt the preparation of lower-charged, cationic liposomes Within our group, cationic liposomes are generally created through mixing DOPE with our cationic lipid CDAN in a 1:1 molar ratio, then extruded and sized (typically, 100 +/- 10 nm (photon correlation spectroscopy (PCS)) Meanwhile, the reporter plasmid (pUMVC1-nt-beta-gal, 7.53 kbp) is precondensed with the adenoviral core peptide mu in a 1:0.6 (w:w) ratio, respectively Complexation of this MD species with cationic liposomes in a 1:12 (w:w) ratio then generates the LMD particle

(140 +/- 10 nm (PCS)) we routinely use for in vitro transfection

studies