Liposomal delivery system for biologically active agents

The present invention is directed to a liposomal preparation which is based on specific lipid components. The liposomal compounds are also combined with a biologically active agent, forming liposomal compounds. These compounds are useful in drug delivery, where specific therapeutic compounds are provided in the liposomes. The specific lipid components of the present invention provide a highly efficient and stable delivery system for nucleic acids. Consequently, one embodiment of the invention provide the liposomal preparations which are suitable for use in gene therapy.

FIELD OF THE INVENTION 
The present invention is directed to a liposomal preparation which is based 
on a composition of specific lipids which form liposomes. It is also an 
object of the present invention to provide a method for preparing a 
liposomal composition carrying a biologically active agent which is simple 
and very efficient. The liposomal delivery system of the present invention 
is used as a highly efficient transfer therapy method. 
BACKGROUND OF THE INVENTION 
Lipidic particles have been shown to be efficient vehicles for many in 
vitro and in vivo applications. Lipidic particles complexed with DNA have 
been used in vitro (Felgner P. L., et al. Proc. Natl. Acad. Sci. USA 84, 
7413-7417 (1987); Gao X. et al. Biochem. Biophys. Res. Commun. 179, 
280-285 (1991)) and in vivo (Nabel E. G., et al. Science 249, 1285-1288 
(1990); Wang C. et al. Proc. Natl. Acad. Sci. USA 84, 7851-7855 (1987); 
Zhu N., et al. Science 261, 209-211 (1993); Soriano P., et al. Proc. Natl. 
Acad. Sci. USA 80, 7128-7131 (1983)) for the expression of a given gene 
through the use of plasmid vectors. Formation of complexes of DNA with 
cationic lipidic particles has recently been the focus of research of many 
laboratories. In particular, lipofectin.TM. (Gibco BRL, Gaithersburg, Md.) 
has been successfully used for the transfection of various cell lines in 
vitro (Felgner P. L., et al. Proc. Natl. Acad. Sci. USA 84, 7413-7417 
(1987)) and for systemic gene expression after intravenous delivery into 
adult mice (Zhu N., et al. Science 261, 209-211 (1993)). 
Lipidic particles may be complexed with virtually any biological material. 
These particles may be complexed with proteins, therapeutic agents, 
chemotherapeutic agents, and nucleic acids and provide a useful delivery 
system for these agents. One such drug delivery system, gene therapy, is 
one such area which has produced promising results. In this area two 
different strategies have emerged: Gene therapy and oligonucleotide-based 
therapeutics. To be successful these two approaches must be mediated by an 
efficient "in vivo" transfer of the nucleic acid material to the target 
cells and there is a need to provide an efficient and safe delivery system 
of nucleic materials. 
Gene therapy may involve the transfer of normal, functional genetic 
material into cells to correct an abnormality due to a defective or 
deficient gene product. Typically, the genetic material to be transferred 
should at least contain the gene to be transferred together with a 
promoter to control the expression of the new gene. 
Viral agents have been demonstrated to be highly efficient vectors for the 
transfection of somatic cells. Retroviruses in particular have received a 
great deal of attention because they not only enter cells efficiently, but 
also provide a mechanism for stable integration into the host genome 
through the provirus. However, clinical use of retroviral vectors is 
hampered by safety issues. A first concern is the possibility of 
generating an infectious wild type virus following a recombination event. 
A second concern is the consequences of the random integration of the 
viral sequence into the genome of the target cell which may lead to 
tumorigenic event. In addition, as retroviruses would only complete their 
life cycle in dividing cells, a retroviral vector would be inefficient in 
targeting cells which are not dividing. DNA viruses such as adenoviruses 
are potential gene carriers but this strategy is limited in the size of 
the foreign DNA adenoviruses can carry and because of the restricted host 
range. However, the advantage of adenoviruses over retroviral vectors is 
their ability to infect post-mitotic cells. 
Synthetic gene-transfer vectors have been subject to intense investigation 
since this strategy appears to be clinically safe. Potential methods of 
gene delivery that could be employed include DNA/protein complexes 
(Cristiano R. J., et al. Proc. Natl. Acad. Sci. USA 90, 2122-2126 (1993)) 
or lipidic particles (Nabel E. G., et al. Science 249, 1285-1288 (1990); 
Felgner P. L., et al. Proc. Natl. Acad. Sci. USA 84, 7413-7417 (1987); 
Wang C. et al. Proc. Nat. Acad. Sci. USA 84, 7851-7855 (1987); Gao X. et 
al. Biochem. Biophys. Res. Commun. 179, 280-285 (1991); Zhu N., et al. 
Science 261, 209-211 (1993); Soriano P., et al. Proc. Natl. Acad. Sci. USA 
80, 7128-7131 (1983)). The genetic material to be delivered to target 
cells by these methods are plasmids. Plasmids are autonomous 
self-replicating extra chromosomal circular DNA. They can be modified to 
contain a promoter and the gene coding for the protein of interest. Such 
plasmids can be expressed in the nucleus of transfected cells in a 
transient manner. In rare events, the plasmids may be integrated or partly 
integrated in the cell host genome and might therefore be stably 
expressed. Episomal plasmid vectors are plasmids able to replicate in the 
nucleus of the transfected cells and may therefore be expressed in a total 
growing cell population. Plasmids have a promising potential considering 
the fact that they may be applied in combination with a synthetic vector 
as carrier and that gene therapy by this means may be safe, durable, and 
used as drug-like therapy. 
Plasmid preparation is simple, quick, safe, and inexpensive representing 
important advantages over retroviral vector strategy. The successful use 
of this genetic tool for "in vivo" approaches to gene therapy will rely on 
the development of an efficient cell delivery system. 
Retroviral vectors have been shown to be very efficient for gene therapy. 
However, their use for in vitro human gene therapy has several 
limitations. Retroviral vectors may, by insertional mutagenesis lead to 
activation of oncogenes and increase the frequency of malignant 
transformation. They will not transfect non dividing cells, and their 
stability and titer are adversely affected by large gene insert. 
Adenoviral vectors which give rise to transient expression are currently 
limited by a demonstrated toxicity in vivo. Presently, 
replication-compromised herpes simplex virus vectors have toxic effects on 
the cells they infect, thus limiting their use for human trials. These 
obstacles have led several laboratories to develop physical means of gene 
transfer such as the pneumatic DNA gun (Yang et al. 1990 Proc. Natl. Acad. 
Sci. USA 9568-72), direct DNA injection (Wolff et al. 1990 Science 
247:1465-68), or liposome delivery vector (Fergner et al. Proc. Natl. 
Acad. Sci. USA 1987 84:7413-17). 
The fact that viral vectors have limitations such as propensity for 
recombination, low titer, and induction of host immunity has initiated 
research into non-viral vectors. The delivery of plasmid DNA via synthetic 
carriers to cells "in vivo" by direct i.v. administration is appealing 
because of its simplicity and potential to reach a far greater number of 
cells than by an "ex vivo" approach. Although the efficiency of "in vivo" 
transfection of DNA plasmids is limited when compared to delivery by viral 
vectors, recent advances, especially in lipidic particle delivery, have 
demonstrated that non-viral gene transfer offers exciting potential, 
including its use in a clinical setting. More recent attempts to deliver 
gene or antisense oligonucleotides has provided a new impetus to lipid 
particle technology (Leonetti, J. P., et al. (1990) Proc. Natl. Acad. Sci. 
USA 87, 2448-2451; Burch, R. M. et al. (1991) J. Clin. Invest. 88, 
1190-1196; Thierry, A. R. et al. (1992) Nucleic Acids Res. 20, 5691-5698; 
Smith, J. G., et al. (1993) Biochim. Biophys. Acta 1154, 327-340). One 
such approach is based on the formation of complexes of DNA with cationic 
lipidic particles. A few therapeutic clinical trial protocols using local 
administration of these complexes are ongoing but data on systemic 
administration is still poorly documented (Wang C. et al. (1987) Proc. 
Natl. Acad. Sci. USA 84, 7851-7855; Zhu, N. et al. (1993) Science 261, 
209-211). 
Several lipids have been used in attempts to prepare liposome-like 
particles. One such lipid mixture is Lipofectin.TM. which is formed with 
the cationic lipid DOTMA, 
N[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl-ammonium chloride, and DOPE, 
dioleylphosphatidyl ethanolamine at a 1:1 molar ratio. The lipidic 
particles prepared with this formulation spontaneously interact with DNA 
through the electrostatic interaction of the negative charges of the 
nucleic acids and the positive charges at the surface of the cationic 
lipidic particles. This DNA/liposome-like complex fuses with tissue 
culture cells and facilitates the delivery of functional DNA into the 
cells (Felgner P. L., et al. Proc. Natl. Acad. Sci. USA 84, 7413-7417 
(1987)). New cationic lipid particles have been developed: 
Lipofectamine.TM. (Gibco BRL), composed of DOSPA, 
2,3-dioleyloxy-N[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium 
trifluoracetate and DOPE at a 1:1 molar ratio. Lipofectace.TM. (Gibco BRL) 
composed of DDAB, dimethyidioctadecylammonium chloride and DOPE at a 1:1 
molar ratio. DOTAP.TM. (Boehringer Mannheim, Ind.) is 1-2-dioleoyloxy-3 
(trimethyl ammonia) propane. 
Behr et al. (Proc. Natl. Acad. Sci. USA 86, 6982-6986 (1989); Barthel F., 
et al. DNA Cell Biol. 12, 6, 553-560 (1993)) have recently reported the 
use of a lipopolyamine (DOGS, Spermine-5-carboxy-glycinediotade-cylamide) 
to transfer DNA to cultured cells. Lipopolyamines are synthesized from a 
natural polyamine spermine chemically linked to a lipid. For example, DOGS 
is made from spermine and dioctadecylamidoglycine (Behr J. P., et al. 
Proc. Natl. Acad. Sci. USA 86, 6982-6986 (1989)). DOGS spontaneously 
condense DNA on a cationic lipid layer and result in the formation of 
nucleolipidic particles. This lipospermine-coated DNA shows high 
transfection efficiency (Barthel F., et al. DNA Cell Biol. 12, 6, 553-560 
(1993)). 
However, the above-described lipid compositions fail to produce liposomes. 
Rather, these investigations synthesized lipid particles, which are 
clusters of lipid molecules which have not formed at least a lipid bilayer 
membrane and therefore also lack an aqueous internal space. Since these 
particles are mere clusters of lipid molecules, the particles lack the 
ability to act as storage units for biologically active agents contained 
therein. 
Therefore it is an object of the present invention to provide a liposomal 
composition capable of carrying internally biologically active agents. 
It is an other object of the present invention to provide an efficient, 
stable and safe liposome-based delivery system for biologically active 
materials. 
Yet another object of the present invention to provide a novel liposomal 
composition comprising a cationic lipopolyamine and a neutral lipid. 
It is yet another object of the present invention to provide a method of 
transferring biologically active agents into cells and patients using the 
instant liposomal delivery system. 
It is a further object of the present invention to provide a method of 
preparing liposomes, useful in providing efficient transfer therapy. 
Yet a further object of the present invention relates to providing a method 
for long-term expression of a gene product from a non-integrated transgene 
in a patient. 
SUMMARY OF THE INVENTION 
The present invention relates to liposome compositions and a method of 
preparing such liposomes. In addition, the present invention relates to 
the administration of the biologically active agent-liposome preparations 
to cells and further to the administration of the liposome preparations to 
patients as a therapeutic agent. 
The liposome compositions of the present invention provide highly efficient 
delivery of biologically active agents to cells. Liposome vesicles are 
prepared from a mixture of a cationic lipopolyamine and a neutral lipid 
and form a bi- or multilamellar membrane structure (referred to herein as 
"DLS-liposomes") A preferred embodiment of the present invention uses a 
spermine-5-carboxy-glycinedioctadecylamide (referred to herein as "DOGS") 
as the cationic lipopolyamine and dioleylphosphatidyl ethanolamine 
(referred to herein as "DOPE") as the neutral lipid. 
The liposomes of the present invention efficaciously deliver biologically 
active agents into the cytoplasmic compartment of human cells. Use of such 
liposomal vehicles make possible high transfection efficiency of 
biologically active materials into cells. 
The present invention also encompasses a method of preparing such a 
liposome composition. The presence of at least one neutral lipid in 
combination with at least one cationic lipopolyamine makes possible the 
formation of liposomes after hydration. According to the method of the 
present invention, liposomes are prepared by mixing together each of a 
cationic lipopolyamine and a neutral lipid in a molar ratio ranging from a 
ratio of 0.02:1 to a ratio of 2:1; evaporating the mixture to dryness; and 
rehydrating. In order to introduce a biologically active agent into the 
liposomes, such agent can be added prior to or after rehydration of the 
dried film. 
In one aspect of the invention, nucleic acids may be associated with the 
liposomes. This association may be accomplished in at least in two ways: 
(1) complex formation between the cationic liposome vesicle and negatively 
charged polyaminon, such as nucleic acid or (2) encapsulation in the 
cationic liposome vesicle. 
The present invention is further directed to a method of treating a subject 
with a suitable pharmaceutical formulation of nucleic acid-liposomes in 
order to deliver specific nucleic acids to target cells of the subject. 
Such a method of treating subjects provides effective delivery of 
oligonucleotides or gene-expressing nucleic acid vectors (e.g. plasmids or 
viral vectors) into cells. Therefore, such a method of drug delivery is 
useful for the transport of nucleic acid based therapeutics. 
Another embodiment of the present invention is directed to a combination of 
a DOGS/DOPE liposome preparation externally anchored through hydrophobic 
interactions with an adenovirus particle. Since adenoviruses enter cells 
via receptor-mediated endocytosis, the combination of adenovirus particles 
and the DLS-liposomes produces an enhanced transduction efficiency. 
Adenovirus particles may also serve as a nucleic acid which is carried in 
the liposome internally. 
The present invention provides a therapeutic method of treating ailments 
and conditions based upon a liposome-facilitated transfer of biologically 
active agents. For example, the present invention provides a 
pharmaceutical liposomal formulation for the delivery of nucleic acids 
using systemic administration to provide long-term expression of a given 
gene. One such method provides direct systematic nucleic acid transfer 
combining the DLS-liposomes with episomally replicative DNA vectors 
carrying the nucleic acid of interest. Alternatively in vitro cell 
transfection followed by tissue transplantation such that the transfected 
cells are incorporated in transplanted tissue. This method is referred to 
as in vitro/ex vivo transfer. Other biologically active agents may be 
encapsulated in the liposomes of the present invention and delivered to 
cells using systemic or in vitro/ex vivo transfer methods.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to the discovery that biologically active 
agents may be associated with liposomes of a specific composition forming 
a stable structure. The liposome containing a biologically active agent 
may then be injected into a mammalian host to effectively deliver its 
contents to a target cell. One such example comprises encapsulating a 
nucleic acid within a liposome and expressing a gene encoded on the 
nucleic acid within the target host cell, through the use of plasmid DNA. 
Conversely the expression of a gene may be inhibited through the use of 
antisense oligonucleotides. Alternatively a chemotherapeutic agent may act 
as the biologically active agent and be encapsulated within a liposome. 
The efficiency of a liposome-mediated drug delivery system is directly 
dependent upon the liposome composition and its resulting association with 
cellular membranes. 
"Biologically active agents" as the term is used herein refers to molecules 
which effect a biological system. These include molecules such as 
proteins, nucleic acids, therapeutic agents, vitamins and their 
derivatives, viral fractions, lipopolysaccharides, bacterial fractions and 
hormones. 
The term "protein" includes any proteaceous material such as peptides, 
protein fragments, protein conjugates, glycoproteins, proteoglycans, 
cytokines, hormones and growth factors. 
The term "therapeutic agents" refers to any drug whose delivery could be 
affected with liposomes. Therapeutic agents of particular interest are 
chemotherapeutic agents, which are used in the treatment and management of 
cancer patients. Such molecules are generally characterized as 
antiproliferative agents, cytotoxic agents and immunosuppressive agents 
and include molecules such as taxol, toxorubicin, daunorubicin, 
vinca-alcaloide, actinomycin and toposites. 
The term "nucleic acids" means any double strand or single strand 
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) of variable length. 
Nucleic acids include sense and anti-sense strands. Nucleic acid analogs 
such as phosphorothioates, phosphoramidates, phosphonates analogs are also 
considered nucleic acids as that terms is used herein. Nucleic acids also 
include chromosomes and chromosomal fragments. 
Antisense oligonucleotides may potentially be designed to specifically 
target genes and consequently inhibit their expression. In addition this 
delivery system may be a suitable carrier for other gene-targeting 
oligonucleotides such as ribozymes, triple helix forming oligonucleotides 
or oligonucleotides exhibiting non-sequence specific binding to a 
particular proteins of other intracellular molecules. For example, the 
genes of interest may include retroviral or viral genes, drug resistance 
genes, oncogenes, genes involved in the inflammatory response, cellular 
adhesion genes, hormone genes, abnormally overexpressed genes involved in 
gene regulation. 
Such nucleic acids may be associated with a liposome composition in 
accordance with this invention. In one embodiment of the present 
invention, modified or unmodified phosphodiester oligonucleotides 
(alternatively referred to as "oligo(dN)") are used as nucleic acids. 
These analogs provide increased nuclease protection and increased cellular 
transport. 
"Liposome" as the term is used herein refers to a closed structure 
comprising of an outer lipid bi- or multi-layer membrane surrounding an 
internal aqueous space. Liposomes can be used to package any biologically 
active agent for delivery to cells. For example, DNA can be packaged into 
liposomes even in the case of plasmids or viral vectors of large size 
which could potentially be maintained in a soluble form. Such liposome 
encapsulated DNA is ideally suited for direct application to in vivo 
systems by a simple intravenous injection. These liposomes may entrap 
compounds varying in polarity and solubility in water and other solvents. 
The liposomes are generally from a bilayer membrane in a uni- or 
multilamellar membranous structure. Generally these liposomes may form 
hexagonal structures, and suspension of multilamellar vesicles. 
The lipid mixture of the present invention comprises a cationic 
lipopolyamine compound. Cationic lipopolyamines useful in the present 
invention include cationic lipid derivatives of polyamines, spermidine and 
spermine as well as others well-known in the art (Theoharides, 1980 Life 
Sci. 27:703-713; Stevens, 1981 Med. Biol. 59:308-313; Morris, Marton Eds., 
Polyamines in Biology & Med., Dekker, N.Y., N.Y., p. 512; U.S. Pat. No. 
5,171,678 and U.S. Pat. No. 5,283,185, all of which are incorporated 
herein by reference). Examples of cationic lipopolyamines include 
2,3-dioleyloxy-N[2sperminecartoxamido)ethyl]-N,N-dimethyl-1-propanaminuim 
trifluoracetate, spermine-5-carboxy-glycinediotadecylamide and 
dipalmitoylphosphatidylethanolamidospermine. A preferred type of 
lipopolyamine comprises a lipid having a quaternary or tertiary amine 
group covalently attached. 
These lipopolyamines can vary in chain length and may be present as 
mixtures of lipopolyamines in the liposome so long as the molar ratio of 
lipopolyamine to neutral lipid is maintained. 
A preferred cationic lipopolyamine is DOGS. 
In order to form stable liposomes, the cationic lipopolyamine is combined 
with a neutral lipid. Such neutral lipids include triglycerides, 
diglycerides and cholesterol and are known in the art, for example as 
described in U.S. Pat. No. 5,438,044 which is incorporated herein by 
reference. In particular a neutral phospholipid is preferred. More 
preferably, the neutral lipid is a neutral amino phospholipid. Most 
preferably, the neutral lipid comprises phosphatidylethanolamine (PE) or a 
derivative of PE such as DOPE (dioleoyl-phosphatidylethanolamine). 
Mixtures of neutral lipids may be used in the liposomes of the present 
invention so long as the molar ratio of lipopolyamine to neutral lipid is 
maintained. 
Liposomes comprising at least one lipopolyamine and at least one neutral 
lipid, present in a molar ratio range of 0.02:1.0 to a ratio of 2.0:1.0 
provide an effective drug delivery system. More preferably, the molar 
ratio of lipopolyamine to neutral lipid is 0.2:1 to a ratio of 0.9:1. For 
optimal transfection efficiency a cationic lipopolyamine/neutral lipid 
molar ratio of about 0.5:1 is used. The liposomal composition of the 
present invention has shown to be stable in a biological environment. In 
the case wherein a nucleic acids is the biologically active agent, it is 
demonstrated that nucleic acids associated with the liposomal carrier are 
completely protected from enzymatic attack, such as from nucleases, and 
that stability in circulating blood after administration may be achieved. 
The liposome delivery system of the present invention comprises a specific 
mixture of lipids. These components are prepared such that a liposome is 
formed and the biologically active agent, such as a nucleic acid is 
contained therein. 
Nucleic acids-based therapeutics are of broad use in therapy of a wide 
variety of diseases and disorders, such as, inherited or acquired genetic 
disease or viral infections. In addition, nucleic acid based therapeutics 
can be used to prevent drug resistance. 
The present invention may utilize one or more nucleic acids or other 
biologically active molecules in conjunction with the liposomal carrier. 
Method of Preparing DLS-Liposomes 
In one embodiment of the present invention, the liposomes are prepared by 
drying a lipid mixture containing a cationic lipopolyamine and neutral 
lipid which are provided preferably in a molar ratio range of 0.02:1 to a 
ratio of 2.0:1. This dried film is then rehydrated. Several methods of 
associating biologically active agents, for example nucleic acids, with 
liposomes are described in this invention. The exemplified embodiment 
comprises hydrating a dried lipid film by introducing an aqueous solution, 
and completely dispersing it by strongly homogenizing the mixture with a 
vortex, magnetic stirrer and/or sonication. Subsequent liposomes are mixed 
with a nucleic acid solution allowing complex formation between positive 
charges of the lipopolyamine-containing liposomes and the negative charges 
of the nucleic acids. Such liposomes are referred to herein as 
DLS-liposome-1 or lipid complexes. 
In another embodiment of the present invention the liposomes are formed by 
dissolving in chloroform at least one cationic lipopolyamine and at least 
one neutral lipid. After stirring by gentle vortexing, the mixture is 
evaporated to dryness. The subsequent dried lipid film is resuspended in a 
volume of water containing the biologically active agent. Formation of 
DLS-liposome is carried out by thorough stirring. Entrapment and/or 
assimilation of the biologically active agent by the DLS-liposomes is 
efficient and nearly complete. 
The exemplified embodiment comprises hydrating the dried lipid film using a 
low and defined (5-10 .lambda.l/.mu.g lipids) volume of aqueous solution 
containing concentrated nucleic acids. These liposomes are referred to 
herein as DLS-liposomes-2 or encapsulated lipsomes. Such concentrated 
nucleic acids are provided in a concentration greater than 1 mg/ml. A 
preferred concentration is about 2 mg/ml and the most concentrated form of 
nucleic acid will depend upon the concentration at which its viscosity is 
excessive, generally at a concentration of about 3 mg/ml. Dispersion is 
completed by strongly homogenizing the mixture using a vortex or magnetic 
stirrer. Nucleic acids are encapsulated in the liposomes during the 
formation and also are partly complexed through electrostatic interaction 
between the nucleic acid and the cationic liposomes. 
In another embodiment of the present invention other biologically active 
agents are encapsulated in a DLS-liposome. The second method described 
above can be used with any biologically active material. Therefore, 
molecules such as chemotherapeutic agents can be introduced into the 
liposomes of the present invention by rehydrating the dried film in the 
presence of such agents. 
The methods of forming liposomes of the present invention lead to 
liposome-complexed and liposome-encapsulated biologically active agents. 
Liposome-encapsulated biologically active agents have been shown to be 
more efficient in transducing cells in cell cultures. However, the ability 
to sonicate the lipid vesicles in the liposome-complexed biologically 
active agents allow for more homogenized and smaller liposome particles, 
and consequently for the ability to circulate for longer periods in blood 
following systemic injection. 
Biologically active agents delivered using the delivery system of the 
present invention are efficiently released from endocytic vesicles, and as 
a result, a high cytoplasmic and nuclear distribution of biologically 
active agents is achieved. 
Targeted Liposomes 
The presence of a neutral lipid, such as DOPE, in combination with a 
cationic lipopolyamine such as DOGS makes possible the formation of 
liposomes upon rehydration, whereas use of a lipopolyamine alone only 
leads to the formation of lipid particles. Formation of phospholipidic 
bilayer or multilamellar membrane vesicles (liposomes) allows for a 
enhanced blood circulation, stability and effectiveness of cellular 
uptake. In addition, the liposomal membrane facilitates anchorage to its 
surface of other substituents, which can increase gene transfer and allow 
cell targeting, such as viral particles, virus fusogenic peptides specific 
ligands or antibodies. 
Formation of liposomes make possible anchorage to the membrane layers of 
products which may increase transduction efficiency. Viruses, in general, 
are inherently excellent gene transfer vectors. Viral capsids or envelopes 
exhibit specific structure and contain molecules leading to efficient 
delivery of their genetic content to the infected cells. In order to 
exploit these properties, adenovirus particles (without DNA or denatured 
by irradiation) have been externally attached to the liposomal membrane of 
the liposomes prepared according to the present invention. It is 
established that adenoviruses enter cells via receptor-mediated 
endocytosis. A specific fusogenic mechanism makes possible the release of 
the viral genetic content from the cellular endocytic vesicles after 
internalization. Use of adenovirus to facilitate gene transfer has been 
reported (Cristiano R. J., et al. Proc. Natl. Acad. Sci. USA 90, 2122-2126 
(1993)). Although DLS-liposomes clearly show a significant escape from 
intracellular vesicles, presence of adenovirus capsids at the liposome 
surface may enhance transduction efficiency by facilitating intracellular 
vesicle disruption. 
Alternatively, tissue targeting may be obtained by anchoring antibodies or 
ligands at the surface of the liposomes. Cell specificity of such liposome 
mediated delivery may be of particular importance in targeting cancer 
cells and bone marrow stem cells. 
The liposomal delivery system of the present invention may be used for 
increasing recombinant retrovirus and adenovirus infection. Retrovirus 
entry into cells is mediated via ligand-receptor recognition, and 
consequently their uptake is very low in certain cells which do not 
present those receptors. Associating a retrovirus or other recombinant 
virus such as adenovirus to be used for gene therapy onto the outside of 
the liposomes may enhance penetration and/or expression of the viral 
agents. 
The liposomal delivery system of the present invention makes possible high 
transduction efficiency in any type of cell, including human 
adenocarcinoma, HeLa, murine carcinoma, NIH3T3, human embryonic kidney 
293, human leukemia MOLT-3 cell lines, and primary cultures of human 
macrophages and human vascular endothelial cells. 
Transfer Therapy Methods 
The liposomal composition of the present invention may be systematically 
administered into patients parenterally in order to achieve transfer 
therapy of one or more biologically active agents. Moreover, this 
technique may be used for "ex vivo" transfer therapy where tissue or cells 
are removed from patients, then treated and finally reimplanted in the 
patient. Alternatively, systemic therapy is also effective in 
administering the DLS-liposome. 
Many diseases can be treated via the drug delivery system of the present 
invention. Diseases such as diabetes, atherosclerosis, 
chemotherapy-induced multi-drug resistance, and generally, immunological, 
neurological and viral diseases can be treated using the present drug 
delivery system. One particular condition which can be treated via the 
system of the present invention relates to HIV and HIV-related diseases, 
such as anemia, leukopenia and thrombocytopenia. These clinical conditions 
are significantly related to a decrease or disappearance of hematopoietic 
progenitor cells in bone marrow of HIV-1 patients. Transfection of bone 
marrow stem cells, bone marrow stroma cells and embryonic stem with gene 
coding for immuno-restoring compounds might enhance the differentiation 
and proliferation capacity of such cells. 
The delivery system of the present invention is also useful for correcting 
the ion transport defect in cystic fibrosis patients by inserting the 
human CFTR (cystic fibrosis transmembrane conductance regulator) gene. 
Oral administration such as nebulization could particularly suitable. In 
addition, DLS-liposomes can be used for the inhibition of tumor cells by 
administering in tumor cells a molecule inhibiting tumorigenesis or gene 
coding for an antisense oligonucleotides directed to mRNA transcripts of 
angiogenic factors. In addition, ribozymes may be encapsulated and 
enzymatically attach specific cellular contents. Intra-lesional or 
intravenous administration appear suitable for this case. 
The ability to select bone marrow cells expressing a selectable gene which 
confers resistance to anti-cancer drugs would be useful to protect bone 
marrow during chemotherapy, but also could be helpful to select for cells 
co-transfected with genes needed in therapy of other diseases, including 
genetic defects manifested in bone marrow. One such selectable gene is the 
human MDR1 gene which confers cross-resistance to many cytotoxic drugs. 
The human MDR1 gene product is a 170 Kd glycoprotein (referred to herein 
as "P-gp") that works as an ATP dependent pump that effectively pumps out 
of cells many anti-cancer cytotoxic drugs, such as topside, teniposide, 
actinomycin D, doxorubicin, daunorubicin, taxol, or vinca alkaloids. 
An exemplified embodiment of the present invention describes an efficient 
protocol for introducing the human MDR1 gene into hematopoietic cells both 
in vivo and in vivo using a liposomal delivery system. Transfection of 
hematopoietic cells followed by gene expression is demonstrated in at 
least three blood cell lineages. 
Using the DLS-liposome system, the human MDR1 gene is introduced into bone 
marrow cells ("BMC"). The transferred human MDR1 is expressed, as detected 
by staining with P-gp specific MRK16 monoclonal antibody, in all of the in 
vitro transfected BMC. Moreover, P-gp is detected in BMC from all 
transplanted animals tested, and from almost all of the in vivo treated 
animals. 
The expression of the MDR1 gene appears to be present for a period of at 
least 30-36 days, indicating that some of the transfected cells had been 
precursor cells, or long-lasting cells. 
The potential for obtaining drug resistant bone marrow progenitor cells 
after gene transfer using the instant liposome delivery system make it 
possible to protect cancer patients undergoing chemotherapy from marrow 
toxicity of anti-cancer drugs. In addition, the multidrug resistance gene 
serves as a positive selectable gene marker in vivo for insuring the 
expression of a non-selectable gene. 
Alternatively a systemic approach to transfer therapy may be utilized. 
The DLS-liposomes containing the nucleic acid drug can be administered by 
intravenous, intramuscular, intraperitoneal, subcutaneous intra-lesional 
and oral means. 
The development of the present liposome delivery system comprising DLS 
liposomes may be encapsulate episomal expression vectors so as to result 
in a broad biodistribution and persistence of transgene expression 
following a single intravenous ("i.v.") injection of liposomal DNA. The 
efficacy of DLS-liposomes used for the "in vivo" expression of the human 
MDR-1 gene is also disclosed in bone marrow progenitor cells by employing 
two different approaches: 1) a systemic delivery, and 2) an "ex vivo" 
approach by transplanting "in vitro" transfected BMC. 
The long-term transgene expression observed using these delivery methods is 
due to the ability of DLS-liposomes to deliver significant amounts of DNA 
in to cells and tissues and to the use of human papovavirus 
("BKV")-derived episomal expression vectors. Episomal vectors may be 
derived from the BKV contain a viral origin of DNA replication and a viral 
early gene that transactivates the viral DNA origin of replication, 
allowing for episomal replication in permissive cells. BKV-derived 
expression vector share the desirable qualities of extrachromosomal 
replication and thus a lack of a requirement of cellular division for the 
chromosomal replication of retroviral-based vectors. Moreover, 
extrachromosomal expression may lessen the possibility of attenuation of 
the transgene expression due to host cis-chromosomal effects. The 
BKV-derived episomes may persist in the progeny of transfected cells, 
whereas non-episomal vectors would not persist in a non-integrated form 
following cell division. Other episomal expression vectors include as 
Epstein-Barr virus derived vectors (Yates, J. L. et al. (1985) Nature 313, 
812-815) and can also be used for "in vivo" gene transfer therapies. 
Episomal expression constructs utilized in the present invention improve 
the persistence of transgene expression when compared with the use of 
non-episomal vectors. Using PCR analysis, detection of the transgene in 
various organs is possible and detection of the transgene mRNA persists 
for as long as 3 months. Transgene expression of the present invention 
declines slowly after a maximum level between 6 and 15 days post-injection 
and is detected for up to 3 months in various tissues. Original and 
episomally replicated forms of BKV-derived derived vectors are present in 
tissues 2 weeks post-injection suggesting episomal replication from this 
point on. 
Episomal DNA vectors in combination with an efficient synthetic delivery 
system appears to be a particularly attractive approach for gene transfer 
therapy. The present invention designed BKV-derived episomal constructs 
which lack the expression of viral proteins (VP1, VP2 and VP3) so as to 
avoid the side effects associated with the expression of viral proteins. 
These constructs (pBKd2) retained high transfection efficiency and "in 
vivo" episomal replication. 
A degree of tissue specific expression can be obtained depending upon the 
liposome preparation, the route of administration and the promoter driving 
expression of the transgene. Useful promoters are well-known to the 
skilled artisan and can be substantiated for those exemplified herein. It 
is clear that the more cationic (DNA/total lipid ratio &lt;0.05, w/w) the 
liposomes, the more lung and heart are targeted. Although reporter gene 
expression may be lower following subcutaneous ("s.c.") administration of 
liposomal DNA compared to i.v. administration, there were no changes in 
tissue targeting. In contrast and as expected, after intraperitoneal 
("i.p.") administration the spleen was particularly targeted. The present 
invention also demonstrates that the CMV promoter is capable of more 
efficient expression in spleen than in lung when compared to the RSV 
promoter. No significant difference has been observed in liver and heart. 
Thus, the choice of the promoter may greatly influence the efficacy of 
non-retroviral mediated gene delivery and may lead to a certain degree of 
tissue specificity. 
In one embodiment of the present invention, the transgene expression 
following a single injection of liposomal DNA was investigated. It is 
clear that repeated injection may increase and/or prolong transgene 
expression. Then, desirable transgenes may be repetitively administrated 
and thus offers an attractive alternatives to retroviral mediated gene 
therapy. Using the DLS-liposomes of the present invention administered via 
systemic delivery or aerosol delivery induced immunogenecity was observed 
when liposomal DNA was administrated at doses which produced detectable 
transgene expression. 
A proposed daily dosage of active compound for the treatment of man is 0.5 
mg DNA/kg to 4 mg DNA/kg, which may be conveniently administered in one or 
two doses. The precise dose employed will of course depend on the age and 
conditions of the patient and on the route of administration. Thus a 
suitable dose for administration by inhalation is 0.5 mg DNA/kg to 2 mg 
DNA/kg, for oral administration is 2 mg DNA/kg to 5 mg DNA/kg, for 
parenteral administration is 2 mg DNA/kg to 4 mg DNA/kg. 
The compound of the invention may be formulated for parenteral 
administration by bolus injection or continuous infusion. Formulation for 
injection may be presented in unit dosage form in ampoules, or in 
multi-dose containers with an added preservative. The compositions may 
take such forms as suspension, solutions or emulsions in oily or aqueous 
vehicles, and may contain formulatory agents such as suspending, 
stabilizing and/or dispersing agents. Alternatively, the active ingredient 
may be in powder form for reconstitution with a suitable vehicle, e.g. 
sterile pyrogen-free water, before use. 
The compounds according to the invention may be formulated for 
administration in any convenient way. The invention therefore includes 
within its scope pharmaceutical compositions comprising at least one 
liposomal compound formulated for use in human or veterinary medicine. 
Such compositions may be presented for use with physiologically acceptable 
carriers or excipients, optionally with supplementary medicinal agents. 
Conventional carriers can also be used with the present invention. 
For oral administration, the pharmaceutical composition may take the form 
of, for example, tablets, capsules, powders, solutions, syrups or 
suspensions prepared by conventional means with acceptable excipients. 
The following examples serve to illustrate further the present invention 
and are not to be construed as limiting its scope in any way. 
All of the references mentioned in the present application are incorporated 
in toto into this application by reference thereto. 
EXAMPLE 1 
Preparation of a DOGS/DOPE liposome composition. 
Liposomes are formed by mixing 1 mg DOGS and 1 mg DOPE (0.5:1 molar ratio). 
After thorough stirring, the mixture is evaporated to dryness in a round 
bottomed borosilicate tube using a rotary evaporator. The subsequent dried 
lipid film is resuspended in a low volume of ethanol (10 to 40 pl/mg 
lipid). Formation of liposomes is carried out by adding an excess of 
distillated water (at least 200 .mu.l/mg lipid). After homogenization by 
slight vortexing, the mixture is incubated for at least 15 min. If needed, 
the resulting suspension may be sonicated in a fixed temperature bath at 
25.degree. C. for 15 min. 
EXAMPLE 2 
Preparation of a DOGS/DOPE liposome-nucleic acids complex composition. 
Complex formation of nucleic acids to the liposome bilayer membrane is 
achieved by simply mixing the preformed DOGS/DOPE liposomes to a solution 
of nucleic acids. In an Eppendorf tube, DLS-liposomes are mixed in a 150 
mM NaCl solution to nucleic acids at a concentration of 12.5 .mu.g total 
lipids (liposomes)/.mu.g nucleic acid for double strand DNA, and a 
concentration of 6 .mu.g liposomes/1 .mu.g nucleic acid for 
oligonucleotides. The mixture is slightly mixed and incubated for at least 
30 min at room temperature. Complex formation is very effective and nearly 
complete since at least 80% of nucleic acids were assimilated into the 
liposomes. These liposomes are referred to as DLS-liposomes-1 or liposome 
complexes. 
EXAMPLE 3 
Alternatively, liposomes were formed by mixing 1 mg DOGS and 1 mg DOPE 
(0.5:1, molar ratio). After thorough stirring, the mixture is evaporated 
to dryness in a round bottomed borosilicate tube using a rotary 
evaporator. The subsequent dried lipid film is resuspended in a minimal 
volume (7 .mu.l/mg lipid) of water solution containing nucleic acids (1400 
.mu.g/ml). Formation of liposomes is carried out by thorough stirring. The 
subsequent liposome preparation may be diluted in 150 mM NaCl. Entrapment 
and/or assimilation of nucleic acid by the liposomes is very efficient and 
nearly complete since at least 80% of nucleic acids are encapsulated by 
the liposomes. These liposomes are referred to as DLS-liposomes-2 or 
encapsulated liposomes. 
EXAMPLE 4 
In vitro Gene Transfection 
Gene transfection efficacy was ascertained in vitro using reporter genes 
such as genes coding for .beta.-galactosidase or luciferase. Two plasmid 
constructs containing the CMV (Clonetech) and RSV (Promega) promoters were 
used as genetic vectors for the .beta.-galactosidase and luciferase genes, 
respectively. These plasmid vectors were delivered to the human carcinoma 
HeLa cells via the liposome carrier system in accordance with this 
invention. Either DLS-liposmes-1 or DLS-liposomes-2 are used in this 
example. 
One microgram liposomal DNA (both DLS-liposmes-1 and 2) was added to the 
medium of a HeLa cell culture at a 50-700% confluency (500,000-700,000 
cells/ml culture medium/7 cm.sup.2 culture plate surface area). Cells were 
incubated at 37.degree. C. with the liposomal DNA for at least 4 hr. 
Determination of gene expression was carried out for both types of 
plasmids following an incubation of 2-3 days at 37.degree. C. 
.beta.-galactosidase activity was observed using a staining procedure after 
cell fixation on the culture plate using a conventional method. Cells 
expressing the .beta.-galactosidase were readily identified by their 
intense blue staining. As shown in FIG. 1, more than 60% of the HeLa cells 
treated by DOGS/DOPE liposomal DNA, actively expressed 
.beta.-galactosidase. 
Luciferase activity was detected in HeLa cells using a standard method 
(Promega, Madison, Wis.). Luciferase gene containing plasmid was used for 
a comparative study of the transduction efficiency of the liposomal 
delivery in accordance with this invention and liposomal vectors 
commercially available. Optimal experimental conditions were used for each 
tested method. 
In serum-containing cell culture medium, transduction efficiency in HeLa 
cells treated with the liposomal system in accordance with this invention 
appears to be 11-fold, 10-fold and 37-fold higher than that of DOGS or 
Transfecta.TM. (Promega, Madison, Wis.), Lipofectin.TM. and 
Lipofectamine.TM. (Gibco BRL, Gaithersburg), as shown in FIG. 2. 
In serum-free medium, transduction efficiency using DLS-liposomes appears 
equivalent to that determined when cells are incubated in serum-containing 
medium. In contrast, use of Lipofectamine.TM. in serum-free conditions 
make possible a high transfection efficiency. The dramatic decrease in 
transduction efficiency (186-fold) using Lipofectamine.TM. in a medium 
containing fetal bovine serum (10%) emphasizes the high instability of DNA 
when exposed to nucleases, and the need for complete DNA protection from 
enzymatic attack in a biological environment. DOGS/DOPE liposomes prepared 
in accordance with the method described in example 2 and 3 exhibit an 
effective cell delivery and an efficient DNA protection during transport. 
As illustrated in FIG. 3, preincubation of liposomal DNA in serum 
containing medium up to 48 hours does not decrease transfection 
efficiency. 
FIG. 4 shows optimal transfection efficiency by using a 12:1 liposome/DNA 
weight ratio for the preparation of the liposomal DNA. 
As illustrated in FIG. 5, use of plasmid DNA delivered by DLS-liposomes-2 
showed better transduction efficiency in HeLa cells when compared to the 
DLS-liposomes-1 delivery. This may be due to increase in DNA protection 
from nuclease attack and/or better release from endocytic vesicles when 
encapsulated in liposomes. 
EXAMPLE 5 
Preparation of DOGS/DOPE Liposome Composition Containing Oligonucleotides 
Oligonucleotides may be complexed or encapsulated in DLS-liposomes using 
the method described in example 2, respectively. The only modification is 
that a two-fold higher nucleic acid/liposome ratio is preferably used for 
DLS-liposomes-1 (20 .mu.g/60 .mu.g, weight ratio) to produce an equivalent 
complexing efficiency. This leads to a higher concentration of 
oligonucleotides complexed with the liposome preparation and consequently 
a higher efficiency of delivery in terms of quantity of nucleic acids 
delivered per cell. 
EXAMPLE 6 
Intracellular Distribution of Nucleic Acid After Delivery with 
DLS-Liposomes 
Nucleic acid cell penetration and its intracellular distribution following 
delivery using DLS-liposomes were observed using laser-assisted confocal 
microscopy and FITC-labeled oligodeoxyribonucleotides (20 mers). The 
former technique allows for the high resolution of optical sections of 
suspension cell preparations and can readily specify the intracellular 
distribution of a fluorescent compound. FIG. 6 presents images of 
hepatocyte HepG2 cells treated with DLS-1 encapsulated 
oligodeoxyribonucleotides for 24 hrs. 
A high penetration of the labeled oligodeoxyribonucleotides was observed in 
all intracellular compartments (FIG. 6A). In order to investigate where 
oligodeoxyribonucleotides are highly concentrated, we significantly 
reduced the gain of the laser beam used for confocal microscopy 
observation of the same cell, and observed a punctuated intra-cytoplasmic 
distribution of the oligonucleotides (FIG. 6B). This suggests that 
DLS-liposomes transport oligonucleotides into cells via endocytosis and 
then oligonucleotides quickly escape from endocytic vesicles leading to a 
release of free oligonucleotides in the cytoplasm. Oligonucleotides are 
immediately transported from the cytoplasm to the nucleus (FIG. 6C). An 
extremely weak fluorescence intensity was observed in cells incubated with 
free labeled oligonucleotides, suggesting poor penetration and/or 
degradation by nucleases present in the serum-containing culture medium 
(FIG. 6D). 
This observation is of great interest since it shows efficient delivery of 
nucleic acids to cells, total and immediate escape from endocytic vesicles 
where active degradation could take place, and nuclear localization after 
cell treatment. Theoretically, cell delivery of plasmid-DNA via 
DLS-liposomes may use the same pathway of cell internalization. 
EXAMPLE 7 
Preparation of a DOGS/DOPE Containing Liposome Composition with Adenovirus 
Particles 
The adenovirus strain used in the present invention is the dl 312 strain 
received as a gift from T. Shenk (Princeton Univ., Princeton, N.J.). Any 
adenovirus strain can be used in the present invention. Preparation of 
adenovirus capsids (no DNA) using cesium chloride gradient method may be 
performed following adenovirus collection and preparation. Whole 
adenovirus particles (with DNA) but inactivated by UV irradiation (10 
J.m.sup.-2 8-1) may also be used. Hydrophobic binding of adenovirus to the 
liposomes is carried out by simply mixing the liposome suspension with the 
adenovirus concentrate. For example, particles equivalent to 10.sup.8 PFU 
(Plating Forming Unit) are added to 12.5 .mu.l of a 
DLS-liposome-associated DNA preparation corresponding to 1 .mu.g DNA and 
12.5 .mu.g lipids. The mixture is slightly homogenized and then incubated 
at 37.degree. C. for 1 hr with gentle shaking. Immediately after 
incubation adenovirus-DNA liposome preparation is added to cell culture. 
Transfection procedure is the same as that used for DLS liposomes. 
FIG. 7 illustrates that adenovirus associated liposomes greatly enhance 
transfection efficiency in HeLa cells by a factor 4.5. Transfection 
efficiency obtained after simultaneous addition in cell culture medium of 
liposomal DNA and adenovirus particles at equivalent concentration was 
significantly lower (2.7-fold). This demonstrates the specific additional 
effect of the adenovirus particle attachment to the liposomes on gene 
expression and particularly on the plasmid DNA escape from endocytic 
vesicles. 
EXAMPLE 8 
Transmission Electron Microscopy Comparison of Lipidic Particles and 
DLS-Liposomes 
DLS-liposomes and Transfectam.TM. (Promega, WI) reagent (DOGS) samples were 
submitted to negative staining and Transmission Electron Microscopy (TEM) 
analysis. The following is a part of the observations independently made 
by ABI Inc (Columbia, Md.). 
DLS-liposomes: "Lipidic particles were observed throughout this preparation 
and were found in large quantities." "Each different particle appeared to 
display a heavily stained core region, which was surrounded by many 
different layers of membranes or envelopes. The particles contained so 
many different layers of membranes, it was difficult to establish the size 
of one lipidic particle to the next. Although it was difficult to measure 
the overall size of the particles, due to their pleomorphic shape and 
varied number of layers, it appeared the particles ranged from 200 to 3000 
nm in diameter. The grid areas showed a high concentration of smaller 
lipidic particles throughout the background of the sample". 
Transfectam.TM. reagent (DOGS): "Possible lipidic particles were found in 
this sample, in small quantities. The particles found in this sample were 
very different from those observed in the previous sample. The lipidic 
particles observed appeared to be either in the process of breaking down 
or they had never properly been formed. Large areas of lipid-like material 
were observed, however, they did not display any ultrastructural detail, 
such as different layers of membranes. The only similarity between this 
sample and the previous sample was that the lipidic particles were heavily 
stained. Very little debris was found in the background of the sample." 
Thus, TEM analysis as demonstrated that DLS-liposomes are bilayer membranes 
vehicles. This specific ultrastructure differentiates DLS-liposomes from 
the Transfectam.TM. reagent or other cationic liposomes thus far 
commercialized, such as Lipofectin.TM. (BRL Co., ND). These nonliposome 
particles, when complexed with DNA do not form membrane bilayer-containing 
vesicles but rather are lipid coating particles that presumably contain 
nucleic acids. Thus they are not be liposomes in the true sense of the 
term. DLS-liposomes provide better efficacy in transferring DNA which can 
be explained by their liposomal structure. Furthermore, we may expect 
improved pharmacokinetic properties such as increased plasmid half life. 
In addition, the presence of a membrane bilayer in DLS-liposomes makes 
possible the anchorage of antibody to their surface which may result in 
cell targeting. 
EXAMPLE 9 
Expression of the MDR-1 Gene in Cultured Murine Bone Marrow Cells 
The MDR-1 gene expresses the P-glycoprotein ("P-gp"), a plasma membrane 
protein involved in the emergence of the Multi-drug Resistance phenotype 
which may occur after chemotherapy. The MDR-1 gene was used in this 
example as a marker of gene delivery in order to assess the efficacy of 
bone marrow transplant of MDR-1 gene transfected bone marrow cells by 
DLS-liposomes, both DLS-1 and DLS-2. 
In order to assess the efficacy of bone marrow transplantation for "ex 
vivo" gene therapy, murine bone marrow cells were transfected with this 
plasmid and the DLS-liposomes and transplanted into Balb-C mice. The 
proliferation and differentiation of transduced hematopoietic progenitor 
cells were detected up to 21 days after transplantation in the spleen and 
the bone marrow, suggesting that the bone marrow transplant had taken 
place. 
Murine bone marrow cells were harvested and quickly transfected with the 
pHaMDR GA plasmid encapsulated in DLS-liposomes. Seven different 
experiments have confirmed that the MDR-1 gene was expressed in bone 
marrow cells since cells continue to grow under selective pressures 
(vincristine). In addition, lymphocyte, macrophage and fibroblast 
populations have been shown to exhibit the MDR phenotype after selection 
(using the rhodamine drug efflux method). 
EXAMPLE 10 
In vitro and in vivo Transfection of BMC 
In order to achieve efficient liposomal transfection, both ex vivo and in 
vivo approaches have been used. The protocols shown in FIG. 1 were 
utilized. In examples 10-13, DLS-liposomes-2 are used. 1) the in vitro/ex 
vivo approach, in which mice were pre-treated with 5-fluoro Uracil 
("5-FU") (150 mg/kg) by the method described in Hodgson et al. (1979 
Nature 281:381-2) were sacrificed, and their bone marrow cells ("BMC") 
were transfected with 10 .mu.g of DLS/MDR in T25 culture flasks (Costar). 
BMC transfected with DLS/Neo was used as negative controls. After 4-5 
days, BMC were transplanted into lethally irradiated mice. Some BMC were 
kept for analysis by FACSort, PCR or kept in suspension culture with or 
without vincristine for 48 hours after which they were tested in semisolid 
medium for their potency to form colonies. 2) The direct in vivo gene 
delivery approach was used, in which 2-3 days after being pre-treatment 
with 5-FU (150 mg/kg), mice were injected intravenously with 75 .mu.g 
DLS/MDR. All negative control mice were injected with DLS/Neo. 
Mice from both groups were sacrificed at different time points, and 
hematopoietic cells were collected for analysis by PCR, FACSort, or 
assayed in methylcellulose for the potential to form colonies in the 
presence of different concentrations of vincristine. 
Mouse peripheral blood (PB) was obtained by eye bleeding. Mouse BMC were 
obtained by flushing the long bones with DMEM using a 21 gauge needle. 
Spleen cells were obtained by pressing the spleens with the barrel of a 3 
cc syringe. 
Bone marrow transplantation ("BMT") procedure 
To assess functional expression of P-gp, 1.times.10.sup.6 transfected cells 
and control cells were incubated in FACSort medium containing 1 .mu.g/ml 
rhodamine 123 (rho123) (Sigma) at 37.degree. C. for 15 minutes. After 
washing, the cells were transferred into rho123 free medium at 37.degree. 
C., and incubated for 3-4 hours. The cells were then washed and analyzed 
by FACSort. Results were displayed as histograms, where efflux of rho123 
would be registered as a decease of fluorescence intensity. 
Mouse Bone Marrow Cell Culture 
Bone marrow cells (BMC) were harvested from 6-12 weeks old C57B1/6 mice 
purchased from Frederick Research Laboratories (Frederick, Md.), and 
housed in a specific pathogen-free environment. Mouse BMC culture was 
carried out in DMEM supplemented with 50 .mu.g/ml penicillin/50 .mu.g/ml 
streptomycin, 2 mM glutamine (Gibco laboratories, Greenbelt, Md.), and 10% 
calf serum (Colorado Serum Company, Denver, Colo.). Cell growth factors 
mouse II3 9100 ng/ml), human II6 (200 ng/ml) (Collaborative Science Inc.), 
and rat SCF (10 .mu.g/ml) (generously provided by Amgen, San Diego, 
Calif.) were added to the media before each experiment. 
Plasmids 
The MDR1 retroviral expression plasmid, pHaMDR1/A containing wild type 
human MDR1 cDNA under transcriptional control of the Harvey Murine Sarcoma 
Virus-Long Terminal Repeat (Ha-MSV-LTR) sequences has been described 
(Pastan, et al. 1985 PNAS 85:4486-90). A plasmid containing the neomycin 
resistance gene wit the same promoter sequence as pHaMDR1/A was used as 
negative control (pHaNeo) (Zhu et al. 1993 Science 261:209-11). 
Liposomes were formed by dissolving in chloroform 1 mg 
spermine-5-carboxy-glycinedioctadecylamid (DOGS) and 1 mg of the neutral 
lipid DOPE (0.5:1, molar ratio) designated further as DLS. After stirring 
by gentle vortexing, the mixture was evaporated to dryness. The subsequent 
dried lipid film was resuspended in a minimal volume (7 ml/mg lipid) of 
water containing plasmid (1.4 mg/ml). Formulation of DLS was carried out 
by thorough stirring. Entrapment and/or assimilation of the plasmid by the 
DLS is efficient and nearly complete. pHaMDR1/A, and pHaNeo plasmids 
entrapped in DLS were identified respectively as DLS/MDR, and DLS/Neo. 
Polymerase Chain Reaction 
Total genomic DNA from in vitro transfected BMC, as well from different 
hematopoietic cells collected from transplanted and in vivo transfected 
mice, was obtained using a DNA extract kit (Gentra systems, Inc., Research 
Triangle Parc, N.C.). The DNA yield and purity were tested by UV 
spectroscopy. PCR was carried out with 1 .mu.g total DNA, 1 unit of 
AmpliTaq Polymerase and reaction kits (Perkins Elmer, Roche, Branchburg, 
N.J.) in a final volume of 100 .mu.l. Each cycle of PCR included a 1 
minute step of denaturation at 95.degree. C., a 1 minute step of primer 
annealing at 57.degree. C., and a 1 minute step of extension/synthesis at 
72.degree. C. The presence of human MDR1 gene specific sequence were 
probed by using a set of primers described in Noonan et al. (1990 PNAS 
87:7160-4) yielding a product size of 167 bp. Each primer was added at 40 
pmol per reaction. PCR products were separated on a 2% agarose gel and 
stained with ethidium bromide. 
Selection of BMC in suspension culture 
3.times.10.sup.6 BMC were added to BMC culture medium containing 30 ng/ml 
vincristine (Sigma). Cells were left for 48 hours in culture after which 
floating cells were collected and pooled with adhering mouse BMC that were 
detached using cell scrappers. Cell were counted on a hematocymeter and 
viability was established by trypan blue exclusion. 
Transfer of MDR1 in gene BMC after transfection using DLS Liposomes 
To assess the transfer of the human MDR1 gene in DLS/MDR transfected murine 
cells, genomic DNAs from different NIH3T3 and hematopoietic cells were 
tested by PCR. Successfully transfected cells gave an amplified product of 
167-bp as shown with positive controls pHaMDR1 (FIG. 8, upper row, lane 
2), NIH3T3 colchicine resistant cells (FIG. 8, upper row, lane 3), and 
NIH3T3 cells transfected with the human MDR1 cDNA using the 
calcium-phosphate precipitation method (FIG. 8, upper row, lane 4). A 
positive band was obtained with in vitro DLS/MDR transfected BMC that were 
selected for 48 hours in 30 ng/ml vincristine (FIG. 8, upper row, lane 7). 
FIG. 8 shows representative positive samples that were obtained from mouse 
hematopoietic tissues. Positive bands were detected in BMC, spleen, and PB 
cells (FIG. 8, upper row, lanes 10, 11, 12 respectively) of an DLS/MDR in 
vivo treated mouse, 15 days after i.v. injection; as well as in BMC and 
spleen cells (FIG. 8, upper row, lanes 13, 14 respectively) of a 
reconstituted mouse, 15 days post-BMT with DLS/MDR1 transfected BMC. The 
MDR1 gene was still detected in BMC 30 days post-transplantation in the 
one mouse tested (FIG. 8, lower row, lane 2). None of the control animals 
DLS/Neo i.v. transfected (FIG. 8, lower row, lane 5, 6, 7; BMC, spleen 
cells, and PB cells respectively), or transplanted with DLS/Neo 
transfected BMC (FIG. 8, lower row, lanes 8, 9: BMC, and spleen cells 
respectively) showed any positive band 15 days post-transfection nor 30 
days post-BMT (FIG. 8, lower row, lane 3: BMC). 
In DLS/MDR in vivo treated mice, specific band for the human MDR1 was 
detected in 5 of 6 BMC samples, 4 of 5 spleen cells, and 4 of 7 PB cells 
tested. BMC from one in vivo treated mouse gave a positive band when 
analyzed 28 days post-i.v. injection with DLS/MDR. In the group of 
transplanted mice, human MDR1 specific band was detected in 3 of 4 BMC, 2 
of 3 spleen cells, and 2 of 2 PB tested cells. All in vitro transfected 
BMC turned out positive for human MDR1 whether before or after being drug 
selected (4 of 4 and 2 of 2 respectively). 
EXAMPLE 11 
In vitro DLS/MDR Transfection Leads to Expression of P-gp in BMC 
Transfection of mouse BMC with DLS/cDNA for 4-5 days did not affect the 
morphology nor the number of cells collected when compared with the 
untransfected BMC control. However, when the cells were subjected to 
selection with 30 ng/ml of vincristine for 48 hours, the numbers of viable 
cells obtained varied greatly. From a total of 3.times.10.sup.6 cells 
plated before the selection pressure was added, only 1.5.times.10.sup.4 
cells, and 2.times.10.sup.4 viable cells were counted in the 
untransfected, and in the DLS/Neo transfected control cells respectively. 
In contrast, 2.times.10.sup.6 cells remained viable in the DLS/MDR 
transfected BMC. Using the human P-gp specific monoclonal antibody MRK16, 
and G2CL monoclonal antibody as an irrelevant negative control antibody, 
FACSort analysis was performed of DLS/MDR transfected BMC not subjected 
(FIG. 9A and subjected to selection with 30 ng/ml of vincristine for 48 
hours (FIG. 9B). The histogram in FIG. 9A shows that after transfection, 
MRK16 monoclonal antibody stained 15% of the cells positively above the 
background fluorescence level. Moreover, the whole histogram representing 
BMC stained with MRK16 monoclonal antibody appeared positively displaced 
when compared to the histogram representing the cells stained with G2CL 
monoclonal antibody. Positive staining was noted each time the analyzed 
was done (5 of 5). 
Using FACSort analysis, we demonstrated the function of the gene product in 
a rho123 efflux assay as early as 5 days post-transfection with DLS/MDR. 
As shown in FIG. 10, 3 hours after exposure to rho123 the fluorescence 
level of the non-selected in vitro DLS/MDR transfected cells was lower 
than that of the control BMC transfected with DLS/Neo. 
Staining for P-gp expression 
Collected cells were resuspended, washed in PBS (Gibco Laboratories) 
supplemented with 0.1% bovine albumin (Sigma) and incubated with MRK16 
(generously provided by Hoecht-Japan), a mouse IgG.sub.2 monoclonal 
antibody specific for an external epitope of the P-gp. As a negative 
control, G2CL (Becton Dickinson, Calif.), a mouse IgG.sub.2a monoclonal 
antibody was used. Non-specific binding to mouse cells were prevented by 
the use of 24G2, a rat anti-mouse Fc receptor monoclonal antibody 
(Pharmingen, San Diego, Calif.) that was incubated for 10 minutes prior to 
the addition of the MRK16 and G2CL. After a 30 minute incubation at 
4.degree. C., the cells were washed and incubated with a secondary goat 
anti-mouse IgG fluoro-iso-thiocyanate conjugated (G.alpha.M IgG-FITC) 
antibody for another 30 minutes at 4.degree. C. After a final wash, the 
cells were analyzed using a FACSort (Becton Dickinson, Calif.), and 
fluorescence intensity levels were illustrated as histograms plotted 
against the X axis on a logarithmic scale, with the relative cell number 
displayed along the Y axis. 
Colony forming unit assay in methylcellulose 
An assay for hematopoietic progenitor cells able to form colonies (CFU-Mix) 
was performed using an established method (Wong et al. 1986 PNAS 
83;3851-4). 1.times.10.sup.4 BMC were plated in semisolid medium 
containing 0.9% methylcellulose (StemCell Technologies Inc., Vancouver, 
Canada), 10% calf serum, 1% serum, 1% glutamine, 1% penicillin, 1% 
streptomycin, 100 ng/ml mouse erythropoietin factor (Sigma), 100 ng/ml 
mouse II3, and 100 ng/ml mouse G-CSF (Pharmingen, San Diego, Calif.). In 
each experiment, all cells were plated in triplicate wells of a 96-well 
microtiter plate (Nunc). CFU-Mix that were clearly expended were 
enumerated using an inverted microscope after incubation at 37.degree. C. 
in a humidified 5% CO.sub.2 atmosphere for 10 to 12 days. 
In vitro transfected BMC, were analyzed for their ability to form colonies 
in semisolid medium in the presence or absence of vincristine. Although 
conditions varied somewhat (these clonogenic assays were done with cells 
from three different in vitro transfection experiments), when no selection 
was applied there were typically between 10 to 18 CFU-Mix per 10.sup.4 
total BMC plated. As in vitro controls, non-transfected or DLS/Neo 
transfected BMC were assayed. FIG. 11, panel A, represent typical colonies 
obtained from DLS/Neo transfected BMC. Their number and morphology was 
comparable to the colonies formed from untransfected and DLS/MDR 
transfected BMC (FIG. 11, panel B). When BMC clonogenic potential was 
tested under selective pressure of 20 ng/ml of vincristine, no colonies 
formed from the untransfected, nor from the DLS/MDR transfected BMC (FIG. 
11, panel D). Table 1, panel A, shows absolute numbers of CFU-Mix obtained 
from 1.times.10.sup.4 plated BMC. 
TABLE 1 
______________________________________ 
normal BMC MDR1 
day 5 in culture 
Neo BMC day 5 
BMC day 5 
A not transfected 
post transfection 
post transfection 
______________________________________ 
no 17.8 .+-. 8.9 
13.2 .+-. 8.3 
12.1 .+-. 4.2 
selection p-0.06 
10 ng/ml 
13.4 .+-. 8.13 
7.7 .+-. 7.1 
9.8 .+-. 3.9 
vincristine 
p = 0.3 
20 ng/ml 
0 
3.6 .+-. 2.6 
vincristine 
0% p = 0.007 
(29.7%) 
______________________________________ 
MDR1 
Neo BMC day 5 
BMC day 5 post 
post transfection, 48 
transfection, 48 
B hours preselection 
hours preselection 
______________________________________ 
no 9.6 .+-. 9.9 
5.4 .+-. 2.1 
39.7 .+-. 9.2 
selection 
p = 0.007 
10 ng/ml 
3.8 .+-. 6.3 2.6 .+-. 1.3 33 .+-. 4.0 
vincristine p = 0.000 
20 ng/ml 
0.6 .+-. 1.1 0.7 .+-. 0.5 26.5 .+-. 7.4 
vincristine 
6.25% 12.9% P = 0.000 
(66.7%) 
______________________________________ 
Under no selection, the number of colonies obtained from the untransfected, 
DLS/Neo transfected, and DLS/MDR transfected BMC were similar, with 
17.8+/-8.8, 13.2+/-8.3, and 12.1+/-4.2 CFU-Mix respectively. When 10 ng/ml 
of vincristine was added to the methylcellulose, only 13.4+/-8.13 CFU-Mix 
were counted from the untransfected, and 7.7+/-7.1 CFU-Mix were counted 
from the DLS/Neo transfected BMC. In contrast, 9.8+/-3.9 (p=0.3) CFU-Mix 
grew from the DLS/MDR transfected BMC. Most significant was the difference 
observed when the BMC were tested under 20 ng/ml of vincristine. No 
colonies grew from the untransfected, nor from the DLS/Neo transfected 
BMC, whereas 3.6+/-2.8 (p=0.007) of the DLS/MDR transfected BMC were able 
to form CFU-Mix. This indicated a efficiency of transfected of 29.70%. 
After transfecting the cells in vitro, the cells were subjected to 30 ng/ml 
vincristine for 48 hours. This significantly enriched for the population 
of MDR1 positive progenitor cells, as shown in Table 1, panel B. Under no 
selection, 39.7+/-9.2 (p-0.007) CFU-Mix were counted from the DLS/MDR 
transfected BMC. Whereas, only 9.6+/-9.9 and 5.4+/-2.1 colonies grew from 
the untransfected BMC, the number of CFU-Mix dropped slightly to 33+/-4.0 
(p=0.000) and 26.5+/-7.4 (p=0.000) when 10 ng/ml or 20 ng/ml of 
vincristine was added respectively. This shows that 66.7% positive cells 
were selected by pre-selecting the transfected cells. In contrast, at 10 
ng/ml vincristine the numbers of CFU-Mix in the untransfected and DLS/Neo 
transfected BMC dropped to 3.8+/-6.3 and 2.6+/-1.3 respectively, and at 20 
ng/ml vincristine the numbers dropped to 0.6+/-1.1 and 0.3+/-0.5 
respectively. 
EXAMPLE 12 
Transplantation of in vitro DLS/MDR transfected BMC and systemic delivery 
of DLS/MDR is followed by P-gp expression in mouse BMC 
Analysis of BMC obtained 5 days post-BMT from recipients of DLS/MDR 
transfected BMC showed no positively stained cells with MRK16, and 
G.alpha.M IgG-FITC antibody. However, 15 days post-BMT as shown on FIG. 
12A, staining of DLS/MDR BMC from a transplanted mouse demonstrated higher 
levels of fluorescence than that of BMC taken from a mouse transplanted 
with DLS/Neo transfected cells, with 19.1% of the cells staining above the 
control levels. P-gp on BMC was still detectable by FACSort analysis 25 
days post-BMT (FIG. 12B), with 21% of the cells staining positively in 
that mouse BMC. 
FIGS. 12C, and 12D represent data obtained by FACSort analysis of BMC from 
two DLS/MDR injected mice stained with MRK16, and G.alpha.M IgG-FITC 12 
days (8 positive out of 9 tested), and 25 days (2 positive out of 2 
tested) post-i.v. injection respectively. At both time points, BMC from 
DLS/MDR transfected mice demonstrated positive specific staining for P-gp 
when compared to the BMC obtained from DLS/Neo transfected mice. At day 
12, DLS/MDR mouse cells stained positively (21.3i) compared to the DLS/Neo 
transfected mice. 25 days post-injection, 14.2% of the BMC stained 
positively. 
EXAMPLE 13 
P-gp expression in different BMC lineages after BMT and systemic delivery 
15 days post-BMT, BMC were harvested from three mice, and analyzed for P-gp 
expression in different cell lineages. BMC were stained with MRK16 and 
G.alpha.M IgG-FITC. FIG. 13, top panel, shows a dot plot fluorescence 
representing cell size (FSC) plotted against cell density (SSC) that 
allowed us to differentiate three morphologically different population of 
BMC. Same cell distribution were seen in the DLS/Neo FIG. 13, (left), and 
the DLS/MDR FIG. 13, (right) transfected BMC: the lymphocytes shown as 
small sized cells of low density (R1), the monocyte as large sized cells 
of intermediate density (R2), and the granulocyte as intermediate sized 
cells of high density (R3). By gating each sub-population, the respective 
MRK16 fluorescence was observed (FIG. 13, lower panel). The FACSort data 
revealed that in each sub-population, namely the lymphocytes, the 
monocyte, and the granulocyte, most of the cells expressed P-gp. 
Similar results as in the transplanted mice were found after analysis of 
BMC obtained from in vivo transfected mice 12 days post-i.v. injection 
(FIG. 14). When populations of BMC were transfected, all three 
hematopoietic cell populations gated, namely the lymphocytes (R1), the 
monocyte (R2), and the granulocyte (R3) specifically stained with MRK 16 
when compared to the background DLS/NEO injected control BMC. 
BMT with DLS/MDR transfected BMC and in vivo treatment with DLS/MDR leads 
to P-gp expression in hematopoietic progenitor cells 
At several time points after reconstitution the presence of drug resistant 
clonogenic progenitor hematopoietic cells were tested (two or three bone 
marrow transplanted mice from each group were tested at each time point). 
The results are presented in Table 2, panel A, representing the means 
values +/- standard deviation of colonies obtained with 20 ng/ml 
vincristine. 
TABLE 2 
__________________________________________________________________________ 
4 days 10 days 
15 days 31 days 
post-BMT post-BMT 
post-BMT post-BMT 
A Neo 
MDR Neo 
MDR Neo MDR Neo 
MDR 
__________________________________________________________________________ 
20 ng/ml 
0 0 
5.8 .+-.+-. 
0 2.5 .+-. 
vincristine 
2.4 
0.7 
p = 0.0000 
p = 0.1 
4 days 10 days 
21 days 36 days 
post-injection 
post-injection 
post-injection 
post-injection 
B Neo 
MDR Neo 
MDR Neo MDR Neo 
MDR 
__________________________________________________________________________ 
20 ng/ml 
0 0 0 13.9 .+-. 
0 7.7 .+-. 
0 2.4 .+-. 
vincristine 3.3 5.0 2.1 
p = 0.1 p = 0.1 p = 0.04 
__________________________________________________________________________ 
With no drug selection added, BMC from DLS/Neo, and DLS/MDR BMT mice 
contained similar numbers of CFU-Mix (i.e.: at day 15 post-BMT, Neo: 
22.7+/10.4, and MDR: 27.3+/-3.5). At 20 ng/ml vincristine, no colonies 
grew on day 4, or day 10 post-BMT from any DLS/Neo, or DLS/MDR recipient 
mice. Still, the results indicate that the transfection efficiency of 8.80 
is real. 
Injected mouse BMC, was also analyzed for its ability to form colonies in 
semisolid medium in the presence or absence of vincristine at different 
time points post-injection. As controls, DLS/Neo injected mice were 
assayed. With no drug selection added, BMC from DLS/Neo, and DLS/MDR 
injected mice contained similar numbers of CFU-Mix (i.e.: at day 21 
post-DLS/MDR and DLS/Neo injection, 47.8+/-21.5, and 59.4+/-24.3 CFU-Mix 
were counted respectively; at day 36 post-injection, 30+/-12.1, and 
28.2+/-6.3 colonies were counted in the DLS/MDR, and DLS/Neo injected mice 
had no CFU-Mix at days 4, 10, 21 and 36 post-treatment respectively. In 
contrast, the DLS/MDR treated mice, that grew no colonies at day 4 
post-injection, had 13.9+/-3.3, 7.7+/-5, 2.4+/-2.1 CFU-Mix, at day 10, 21, 
and 36 post-injection respectively. The difference was significant (p, 
&lt;0.05) only for the value taken at day 36, indicated an actual 
transfection efficiency of 8%. 
EXAMPLE 14 
In vivo administration of DLS-liposomes into Balb-C mice. 
Plasmid DNA. Firefly "Photinus pyralis" peroxisomal luciferase gene (luc) 
was used as a report gene for the monitoring of transgene expression 
levels. In preliminary studies episomal vectors were constructed 
containing a genomic fragment of the human papovavirus, BKV. This fragment 
included the BKV viral early region and origin of replication, the large T 
antigen, and later viral capsid proteins. Sequence encoding the firefly 
luciferase (luc; 1.7 kb Bam HI-Sal I fragment of PGEM-luc [Promega]) gene 
was inserted under the control of the Rous sarcoma virus (RSV) promoter 
and the polyadenylation signal and transcriptional termination sequences 
from SV40. The resultant episomal/reporter expression vector was termed 
pBKd1RSv-luc. For subsequent studies, a modified BKV plasmid containing 
the luc gene was constructed in which the entire sequences coding for the 
late BKV capsid proteins (VP1, VP2 and VP3) (Seif, I. et al. (1979) Cell 
18, 963-977) were deleted to remove the expression of these potentially 
immunogenic proteins. The resultant episomal/reporter expression vector 
was termed pBKd2RSV-luc. In addition, a second series of pBKd1 and pBKd2 
vectors were made in which luc expression was placed under the control of 
the enhancer/promoter sequences from the immediate early gene of the human 
cytomegalovirus (CMV) and the polyadenylation signal and transcriptional 
termination sequences from the bovine growth hormone gene. The resultant 
episomal/reporter expression vectors were termed pBKd1CMV-luc and 
pBKd2CMV-luc, respectively. The non-episomal pRSV-luc containing luc under 
the control of the RSV long terminal repeat was constructed as previously 
reported (DeWett, J. et al. (1987) Mol. Cell. Biol. 7, 725-737). 
pCMVintlux plasmid encodes the luc under the control of human CMV 
immediate-early promoter with intron A (Manthorpe, M. et al (1993) Hum. 
Gene Ther. 4, 419-431). 
Preparation of the DLS Liposomes. Liposomes were formed by mixing 1 mg 
dioctadecyl-amidoglycyl spermidine and 1 mg dioleoyl-phosphatidyl 
ethanolamine. After thorough stirring, the mixture was evaporated to 
dryness in a round-bottomed borosilicate tube using a rotary vortex 
evaporator under vacuum. Then the dry lipid film was hydrated with a 
maximum volume (60 .mu.l/mg lipid) of a solution containing 160 .mu.g 
plasmid DNA and was slightly vortexed. After incubation at room 
temperature for 15 min, the resulting suspension was vigorously mixed by 
vortex. Subsequent liposomal DNA preparation was then diluted in 150 mM 
NaCl, and kept at 4.degree. C. Liposomes appeared to range from 200 to 
3,000 nm in diameter as determined by transmission electronic microscopy. 
DLS liposomes consist of multilamellar bilayers vesicles which may complex 
as well as encapsulate DNA. The entrapment rate was found to be 88.+-.8% 
(mean .+-.S.D.) of the initial DNA input dose. This type of liposomes were 
used in examples 14-17. 
In Vivo Gene Transfer. Plasmid-DNA in DLS-liposomes-2 liposome was 
administered by a single injection of 100 to 600 .mu.l total volume in the 
tail vein of 4-6 week old female Balb/C mice. Control mice received 150 mM 
NaCl solution. Mouse tissues were collected in 2 ml Eppendorf tubes, 
quickly frozen on dry ice and stored at -70.degree. C. until examined. 
Plasmid vector containing the luciferase gene as a marker gene were 
delivered by DLS-liposomes in Balb-C mice. Various formulations of 
liposomes encapsulated plasmid at various DNA/lipid ratios were assayed. 
In these experiments transgene expression has been assayed in liver, lung 
and spleen. Luciferase activity was determined by bioluminescence 
measurement (2-3 mice/point). More than 100 mice have been studied. PCR 
analysis showed the long lasting expression of the lucriferase gene in all 
tissues tested (lung, liver, heart, spleen, skeletal muscle, blood cells, 
bone marrow, and ovary) up to at least 2 months post-injection. Only 
episomal replicating DNA vectors showed positive results. 
EXAMPLE 15 
Intravenous administration of DLS-liposomes containing plasmid DNA: Dose 
Response Study 
DNA Dose-Response. To assess for treatment-related toxicity and to 
determine the optimal liposomal DNA amount to be injected, dose-response 
experiments were carried out. Episomally replicative pBKd1RSV-luc plasmid 
DNA encapsulated in DLS liposomes was administered i.v. into mice. 
Luciferase activity was determined 4 days post-injection in liver, lung, 
and spleen (FIG. 15). Luciferase activity could be detected in these 
tissues even when the amount of injected DNA amount was as low as 25 
.mu.g/mouse. No significant difference in luc gene expression between 
tissues was observed at this concentration while at 100 .mu.g DNA 
injected/mouse a significant difference in activity was observed between 
lung, liver and spleen (1700.+-.100, 1020.+-.300 and 490.+-.130 fg 
luciferase/mg protein, respectively; mean .+-.S.D.). In contrast to lung 
and spleen where the transgene expression reached a plateau at 
approximately 200 .mu.g and 50 .mu.g DNA, respectively, there was a 
decrease in transgene expression in the liver with DNA deliveries greater 
than 100 .mu.g, perhaps due to toxicity of the liposomal DNA in liver 
cells or a greater accumulation of liposomal DNA in this organ. No 
luciferase activity could be detected in liver or lung tissue of mice 
injected with 100 .mu.g "naked" pBKd1RSV-luc plasmid or the pBKd1RSV 
plasmid construct containing the lacz gene instead of luc presented in DLS 
liposomes. Macroscopically slight toxicity was observed (enlarged spleen 
and pale liver) when 100-500 .mu.g of liposomal DNA were injected into 
mice. Marked toxicity (grayish lung, tissue damage in heart and occasional 
death) was observed when more than 150 .mu.g liposomal DNA were 
administered. No gross or microscopic anatomical pathology was observed at 
liposomal DNA doses &lt;100 .mu.g. The DNA/lipid ratio was critical as an 
increase in positively charged lipids may contribute to serious toxicity. 
The efficiency of DLS liposomes to deliver the transgene depended directly 
upon the cationic lipid amount in the preparation. Transgene expression in 
lung greatly increased from 55.+-.20 to 3750.+-.950 fg luciferase/mg 
protein (mean .+-.S.D.) when the DNA/lipid ration (w/w) decreased from 
0.32 to 0.08. A ratio of 0.08 was optimal and was used throughout this 
work. 
EXAMPLE 16 
Effect of the Route of Administration. pBKd1RSV-luc plasmid DNA (25 .mu.g) 
were injected intravenously (i.v.) (tail vein), Intraperitoneally (i.p.) 
and subcutaneously (s.c.) and luciferase activity was measured 4 days 
post-injection. No significant difference was observed in the spleen by 
use of different routes of administration but slightly more activity was 
detected in the liver and lung with the i.v. route (FIG. 16). S.c. 
injection appeared to be the less efficient route of administration. I.p. 
administration resulted in a preferential targeting to the spleen. 
Immunohistochemical Detection of Luciferase Expression. Luciferase 
expression was detected in tissue samples from mice treated with 75 .mu.g 
pBKd2CMV-luc and sacrificed 6 days post-injection by immunohistochemical 
staining using a polyclonal antibody against luciferase protein (FIG. 17). 
The recombinant protein was detected in all tissues tested although the 
percentage of positive cells varied: heart, &gt;75%; spleen and liver, &gt;50%; 
and colon and lung &gt;10%. The pattern of transgene expression was 
generalized throughout the heart (FIG. 17A, vascular endothelial cells and 
myocytes), the liver (FIG. 17B, hepatocytes, Kupfer cells and endothelial 
cells) and the spleen. Although staining intensity was lower in the colon 
(FIG. 17D) and in lung, staining was also diffuse in these tissues. In 
separate experiments, 50 pg pBKd1RSV-luc in DLS liposomes were injected 
i.v. in immunodeficient mice bearing Kaposi's sarcoma (KS Y-1) tumor cells 
(Lunardi-Iskandar, Y. et al. (1995) Nature 375, 64-68). The luciferase 
protein was detected in tumor by immunostaining with more than 10% of the 
tumor cells being positive (FIG. 17E). No positive cells were found in 
liver (FIG. 17C) or KS Y-1 tumor cells (FIG. 17F) when luciferase antibody 
was replaced by normal rabbit serum as a control. 
EXAMPLE 17 
Stability of Luciferase Expression in Different Tissues. pBKd2CMV-luc 
plasmid DNA (75 .mu.g) was administered i.v. and luciferase activity was 
measured in liver, lung, spleen and heart at different times (FIG. 18). 
TABLE 3 
__________________________________________________________________________ 
Detection of the luciferase gene in mouse tissues by PCR analyses 
PCR Liver 
Lung 
Spleen 
Heart 
Muscle 
BMC 
PB Brain 
Ovary 
__________________________________________________________________________ 
6 days 
2/2 2/2 
2/2 2/2 
3/3 3/3 
3/3 ND 3/3 
2 weeks 
ND 
ND 
ND 
ND 
2/2 
2/2 
ND 2/2 
2/2 
1 month 
2/2 
1/2 
1/2 
1/2 
2/2 
1/2 
1/2 
2/2 
1/2 
2 months 
2/2 
2/2 
1/2 
1/2 
2/2 
2/2 
1/2 
0/2 
0/2 
3 months 
1/1 
1/1 
1/1 
1/1 
0/1 
0/1 
0/1 
0/1 
0/2 
6 months 
0/2 
0/2 
0/2 
0/2 
0/2 
0/2 
0/2 
0/2 
ND 
__________________________________________________________________________ 
Number of positive tissues/Number of treated mice. 
75 .mu.g of DLSpBKd2CMV were administered i.v. in mice. 
BMC, bone marrow cells; PB, peripheral blood; ND, not determined. 
Transgene expression was maximal between 6 and 15 days post-injection in 
lung, spleen and heart and then gradually declined over 3 months. 
Luciferase activity was low and constant for up to 3 months in the liver. 
The luciferase activity was also detected at 6 days in other organs such 
as the skeleton muscle, brain, bone marrow cells (BMC) and peripheral 
blood (PB) (398.+-.130, 192.+-.103, 2220.+-.73 and 160.+-.46 fg/mg 
protein, respectively; mean .+-.S.D.). Peripheral blood mononuclear cells 
obtained from whole blood after ficoll purification contained 900 fg/mg 
protein which corresponded to approximately 1000 detected molecules of 
luciferase/cell. 
PCR analysis confirmed the presence of luciferase activity since all tested 
tissues were positive for luc 6 days post-injection (Table 3). The 
transgene was found for up to 1 month in brain and ovary, for up to 2 
months in muscle, BMC and PB, and for up to 3 months in lung, liver, 
spleen and heart. In addition, kidney and colon tissues were positive 2 
months post-injection. 
Luc expression was detected by RT-PCR analysis. As shown in FIG. 19, liver, 
spleen lung were positive 3 months post-injection. 
Effect of Plasmid Construct and Promoter on Transgene Expression. The 
expression of six luciferase plasmids in different organs were compared 
beginning 6 days after a single i.v. injection of 75 .mu.g DNA delivered 
with DLS liposomes. The results at 6 days are summarized in Table 4 and 
show that when using the pRSV-luc plasmid luciferase activity was only 
detected in lung. In contrast, luciferase activity was high in all tissues 
tested (liver, lung, spleen and heart) when pCMVintlux was used. The four 
BKV-derived episomal plasmids showed slightly less or equivalent reporter 
gene activity. The CMV promoter yielded significantly higher levels of 
gene expression in spleen and lower levels in lung when compared with the 
RSV promoter driving the same plasmid DNA construct. 
TABLE 4 
______________________________________ 
Plasmid construct and promoter effects on transgene expression 
Liver Lung Spleen Heart 
______________________________________ 
pRSV-luc 0 ++ 0 0 
pCMVintlux ++ +++ 
++ 
+++ 
pBKd1RSV-luc + +++ ++ 
pBKd1CMV-luc ++ +* +++.sup..dagger. 
+++ 
pBKd2RSV-luc ++ +++ + ++ 
pBKd2CMV-luc + ++.sup..dagger-dbl. 
+++.sup..sctn. 
++ 
______________________________________ 
Relative luciferase activity: 0, not detectable; +, 0-0.1 pg/mg protein; 
++, 0.1-1.0 pg/mg protein; +++, higher than 1.0 pg/mg protein. i.v. 
administration, activity determined 6 days postinjection. pBKd1CMVluc 
versus pBKd1RSVluc in *lung (p = 0.011) and in .sup..dagger. spleen (p = 
0.001), and pBKd2CMVluc versus pBKd2RSVluc in .sup.555 lung (p = 0.187) 
and in .sup.517 spleen (p = 0.021). 
The time course of luc expression in mouse tissues from the non-replicative 
pCMVintlux and episomal pBKd2CMV-luc plasmid constructs was followed after 
a single i.v. injection of 75 Ag DNA (FIG. 17). Luc product expressed from 
pBKd2CMV-luc was detected in lung, liver, spleen and heart for up to 2-3 
months post-injection. No detection of luciferase activity was observed in 
these tissues 1 month after injection of pCMVintlux. 
EXAMPLE 18 
Intraperitoneal administration of DLS-liposomes containing DNA. 
Luciferase gene expression was detected in spleen 3 days post-injection. 
The dose injected per mouse was 100 ug. No toxicity was detected. 
EXAMPLE 19 
Inhibition of KS Y-1 cell tumorigenicity. 
The present invention can be used in the therapy of Kaposi's Sarcoma 
("KS"). Two KS cell lines, showing tumorigenic properties in vitro and in 
vivo, have recently been established. One cell line, KS Y-1, was derived 
from a lesion of an HIV-infected individual. The second cell line, KS 
N1506, was derived from a lesion of a non-HIV associated immunodepressed 
individual. High amounts of IL-6, IL-8, and VEGF are produced in these 
cell lines. Correspondingly, high levels of these cytokines have also been 
found in the serum of AIDS-KS patients. In this example, antisense 
oligo(dN) was used as a specific molecular tool to inhibit KS cell 
production of these factors. 
0.1 uM VEGF antisense phosphodiester oligodeoxynucleotides encapsulated in 
DLS-liposomes completely blocked KS Y-1 cell colony formation in 
semi-solid culture. Lipofectin.TM.liposomes required 7-10 fold higher 
concentration to achieve the same inhibitory effect.