Gene Delivery to Mammalian Cells: Volume 1: Nonviral Gene Transfer Techniques

295 Pages·2004·2.74 MB·English

by  Dexi Liu, Evelyn F. Chia, Hui Tian (auth.), William C. Heiser (eds.)

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1
Chemical Methods for DNA Delivery
An Overview
Dexi Liu, Evelyn F. Chiao, and Hui Tian
1. Introduction
Introduction of DNAinto mammalian cells is a powerful tool for studying the
function of various DNAsequences, and for gene therapy. The process of intro-
ducing DNAinto cells for the purpose of gene expression is called transfection
or gene delivery. Synthetic compounds used to facilitate DNAtransfer are often
named synthetic vectors or transfection reagents. Compared with biological
(viral vectors) and physical methods that are covered elsewhere in this volume
and in the next volume, the major advantages of synthetic vectors (or chemical
methods) are their simplicity, ease of production, and relatively low toxicity.
Many synthetic compounds have been developed since DEAE-dextran was first
used in transfection experiments more than 35 years ago. Rapid progress in de-
veloping more efficient synthetic vectors has led to successful DNA delivery
into a variety of cell types in vitro and in vivo. More importantly, in the last few
years, we have witnessed significant efforts and progress in elucidating the
mechanisms underlying synthetic vector-mediated DNAdelivery. With the con-
tinuous effort to meet the need for safe and efficient gene-delivery methods for
human gene therapy, it is foreseeable that significant advances will be made in
the future. This article concentrates on four major types of chemical reagents that
are available to most investigators: calcium phosphate, DEAE-dextran, cationic
lipid, and cationic polymer. Each of these types of reagents has its advantages
and disadvantages, some of which we briefly outline in this overview chapter.
From:Methods in Molecular Biology, vol. 245:
Gene Delivery to Mammalian Cells: Vol. 1: Nonviral Gene Transfer Techniques
Edited by: W. C. Heiser © Humana Press Inc., Totowa, NJ
3
4 Liu et al.
One type of compound that is not covered in this section is biopolymers.
These compounds, including polylysine (1–7), histone (8,9), chitosan (10,11),
and peptides (12–17), have shown relatively low transfection efficiency when
used alone. Although they might become relatively more important in the fu-
ture, we feel their utility has not, at present, been demonstrated to be broad
enough to recommend their routine use for transfection. For those who are in-
terested in obtaining more information about the properties and activity of this
group of polymers in DNAdelivery, relevant information can be found in the
references cited at the end of this chapter or in relevant chapters in this volume.
2. For What Purpose Are Synthetic Vectors Needed?
Cell membranes are sheetlike assemblies of amphipathic molecules that sep-
arate cells from their environment and form the boundaries of different or-
ganelles inside the cells. However, these physical structures, allow only the con-
trolled exchange of materials among the different parts of a cell and with its
immediate surroundings. DNA, on the other hand, is an anionic polymer, large
in molecular weight, hydrophilic, and sensitive to nucleases in biological ma-
trices. Unless special means are used, DNAmolecules are not able to cross the
physical barrier of membrane and enter the cells. In theory, for a successful in-
troduction of DNAinto cells and expression of the encoded gene, one needs to
overcome three major hurdles. These include: (1) transfer of DNAfrom the site
of DNAadministration to the surface of target cells; (2) transfer of DNAacross
the plasma membrane into the cytoplasm; and (3) transfer of the DNAacross
the nuclear membrane into the nucleus to initiate gene expression. Thus, the
corresponding properties for an ideal synthetic vector should include protecting
DNAagainst nuclease degradation; transporting DNAto the target cells; facil-
itating transport of DNA across the plasma membrane; and finally promoting
the import of DNAinto the nucleus.
3. Properties of the Synthetic Compounds Most Commonly Used
for DNADelivery
3.1. Calcium Phosphate
Transfection with calcium phosphate was developed by Graham and Van der
Eb in 1973 (18)and has been one of the most commonly used methods for DNA
delivery into mammalian cells. This method takes advantage of the formation
of small, insoluble, calcium-phosphate-DNAprecipitates that can be adsorbed
onto the cell surface and be taken up by cells through endocytosis. The proce-
dure requires mixing of DNAwith calcium ions, subsequent addition of phos-
phate to the mixture, and presentation of the final solution to cells in culture.
Various types of cells have been transfected using this procedure. Transfection
Chemical Methods for DNADelivery 5
efficiency can be as high as 50%, depending on the cell type and the size and
quality of the precipitate. The variation in the composition and particle size of
the calcium-phosphate-DNA precipitates results in poor reproducibility. This
method does not seem to work on cells in primary culture or in animals.
3.2. DEAE-Dextran
DEAE-dextran was the very first chemical reagent used for DNAdelivery. It
was initially reported by Vaheri and Pagano in 1965 (19)for enhancing the viral
infectivity of cells. Similar to cationic polymers (seeSubheading 3.4), DNAand
DEAE-dextran form aggregates through electrostatic interaction. Aslight excess
of DEAE-dextran in the mixture results in net positive charge in the DEAE-dex-
tran/DNAcomplexes formed. These complexes, when added to cells, bind to the
negatively charged plasma membrane and then are internalized through endocy-
tosis. Compared to calcium phosphate, the transfection efficiency of DEAE-dex-
tran is much higher, although it varies with the type of cells and other experi-
mental conditions. Transfection efficiency as high as 80% has been reported
with DEAE-dextran/DNAcomplexes. The method is relatively simple and re-
producible, but requires low or no serum during transfection. DEAE-dextran is
toxic to cells at high concentration.
3.3. Cationic Lipids
The use of cationic lipids for DNAdelivery was pioneered by Felgner and col-
leagues (20). The first cationic lipid synthesized for the purpose of DNAdelivery
was N-(2,3-dioleyloxypropyl) N, N, N-trimethylamonium chloride (DOTMA).
When hydrated, DOTMAforms liposomes with or without additional lipid com-
ponents. When mixed with DNA, the positive charges at the liposome surface
electrostatically interact with the negative charges on the phosphate backbone of
the DNAto form DNA/liposome complexes (lipoplexes). Addition of lipoplexes
to cells in culture normally results in significant levels of gene expression, with an
efficiency ranging from 5% to more than 90% depending on the type of cell line
used. Abroad spectrum of transfection activity among many cell types, low toxi-
city, and high efficiency are the major advantages of cationic lipids.
Major efforts have been made over the past decade to optimize the transfec-
tion activity of cationic lipids. Results from in vitro and in vivo studies have re-
vealed that, among the many physicochemical properties that affect transfection
activity of cationic liposomes, the most important one is the cationic lipid struc-
ture. The common features shared by all transfection-active cationic lipids de-
veloped so far include three structural domains: (1) a positively charged head
group, (2) a hydrophobic anchor, and (3) a linker connecting the head group and
the hydrophobic anchor. The structure of the head group varies from one to four
ammonium groups, which can be primary, secondary, tertiary, or quaternary for
6 Liu et al.
multivalent cationic lipids (21,22). The most common structure for the hy-
drophobic anchor consists of either two hydrocarbon chains or cholesterol. With
a few exceptions, glycerol is the linker in cationic lipids with a double-chain an-
chor, whereas a variety of linkers have been used in cationic lipids with a cho-
lesterol anchor (21,22). Our studies on structure–function relationships for in-
travenous transfection revealed that the optimal lipid structure should include
the following: (1) a cationic head group and its neighboring hydrocarbon chain
being in a 1,2-relationship on the backbone, (2) an ether bond to link the hy-
drocarbon chain to the backbone, and (3) paired oleyl chains as the hydropho-
bic anchor (25). Cationic lipids without these structural features had lower in-
travenous transfection activity in mice.
For in vitro transfection, structure-based rules that allow for prediction of
transfection activity of cationic lipids have yet to be established. The transfection
activity of cationic lipids varies significantly with the type of cells used. Inclusion
of specific lipids (helper lipids) such as dioleoylphosphatidylethanolamine
(DOPE) into cationic liposomes enhances transfection activity in some cell types
but not others (23). For transfection of epithelial cells by airway administration,
Lee et al. have shown that cholesterol-based cationic lipids (lipid-67) exhibit
much higher activity than their noncholesterol-based counterparts (24). The
mechanism of lipid-67-based gene transfer into lung epithelial cells is not known.
Although many complex structures between DNA and cationic liposomes
have been identified (26–29), the most important parameters affecting the trans-
fection activity of cationic liposomes appear to be the particle size of the com-
plexes (30) and the charge ratio (amines to DNA phosphates ratio, (cid:1)/(cid:2))
(20–25). Complexes with larger particle size ((cid:3)200 nm) and with charge ratio
of slightly greater than 1 appear to be optimal for in vitro transfection. For in-
travenous transfection, however, a charge ratio ((cid:1)/(cid:2)) of greater than 12 is re-
quired for an optimal transfection activity (31,32). Cholesterol generally func-
tions as a better helper lipid than DOPE for systemic transfection (31,33,34).
3.4. Cationic Polymers
Different from the hydrophobic cationic lipids, cationic polymers are a group
of highly water-soluble molecules. Three general types of cationic polymers have
been used for transfection: linear (polylysine, spermine, and histone), branched,
and spherical. The most extensively studied cationic polymers for DNAdelivery
are polyethyleneimine (PEI) (35,36) and dendrimers (37–39). PEIs are highly
branched organic polymers produced by polymerizing aziridine (40). In princi-
ple, PEI, with every third atom in the polymer being amino nitrogen, is a type of
organic compound that contains the highest density of potential positive charges.
It has been estimated that about 25, 50, and 25% of nitrogens in branched PEIs are
primary, secondary, and tertiary amines, respectively. The enriched nitrogen
Chemical Methods for DNADelivery 7
atoms and the diversified nitrogen forms provide considerable buffer capacity for
the PEIs over a wide pH range (41,42). Both branched and linear types of PEIs
have been used for transfection. The in vitro transfection efficiency of PEI 800
KDa on a large variety of cell lines and primary culture is comparable to that of
cationic lipids.
Dendrimers are a new class of branched, spherical, and starburst molecules.
Dendrimers differ in their initiator structure and in the number of layers of the
building blocks in each molecule. (The number of layers is also called the num-
ber of generations.) The common initiators include ammonia (NH ) as trivalent
3
initiator and ethylenediamine as a tetravalent initiator. Polymerization takes
place in a geometrically outward fashion, resulting in branched polymer with
spherical geometry and containing interior tertiary and exterior primary amines.
The defined structure and large number of surface amino groups of dendrimers
have led to these polymers being employed as a carrier for DNAdelivery. Dif-
ferent forms of dendrimers have been shown to be active in transfection (37–39).
Their precisely controlled size and shape provide them with the potential advan-
tage of forming more homogeneous and highly reproducible DNAcomplexes.
When mixed with DNA, cationic polymers readily self-assemble with DNA
and generate small tortoidal or spherical structures of approx 40–100 nm (42),
depending on polymer size, structure, DNAto polymer ratio, and the type and
concentration of ions in the buffer. The complexes formed between DNAand
cationic polymers are called polyplexes. When added to cells in culture, poly-
plexes are taken up by the cells. Similar to that of cationic lipids, transfection
activity of cationic polymers varies with cell type, structure, and size of the
polymer, and polymer to DNA ratio. Compared to cationic lipids, the major
drawback of cationic polymers is their relatively high toxicity.
3.5. Combined Systems
Transfection activity of combined synthetic compounds has been explored.
Depending on the transfection reagents selected, a significant enhancement in
transfection activity can be achieved. In fact, much higher transfection activity
of cationic liposomes was reported when mixed with polylysine (43), protamine
sulfate (44), peptides (45,46), or PNA(Chapter 6 in this volume). The mecha-
nisms for such synergistic effect between cationic liposomes and polymers are
not known, but it is believed that the structure of DNAcomplexes in the com-
bined system may be more effective in escaping the endosomal degradation
and/or more efficient in facilitating DNAtransfer into the nucleus.
4. Mechanisms of DNADelivery by Synthetic Vectors
Acentral tenet of DNAdelivery by synthetic compounds is that DNAmole-
cules can only be delivered into a cell when they are converted into a particu-
8 Liu et al.
late form. This appears to be true for all of the synthetic compounds that have
been developed for transfection thus far. Obviously, synthetic compounds such
as calcium phosphate, DEAE-dextran, cationic lipids, and cationic polymers are
all capable of forming particles with DNA. With the exception of the calcium
phosphate method that forms calcium-phosphate-DNAprecipitates, other syn-
thetic compounds form complexes with DNAthrough electrostatic interaction
between DNAand the synthetic vectors. Once complexed with a synthetic vec-
tor, DNA molecules are protected against nuclease-mediated degradation. To
accomplish DNAdelivery, these complexes need to (1) bind to the cell surface,
(2) cross the plasma membrane, (3) release DNAinto the cytoplasm, and finally,
(4) transport the DNAinto the nucleus. The following is a brief summary of the
current understanding on these events as they are involved in synthetic com-
pound-mediated DNAdelivery.
4.1. Binding to the Cell Surface
Without exception, binding of DNAcomplexes to the cell surface with the
most commonly used synthetic compounds is accomplished by electrostatic in-
teraction between the positively charged complexes and the negatively charged
cell surface. It has been shown that cell surface binding of DNAcomplexes can
be significantly enhanced by centrifugation of the transfection reagents onto cell
surface (47). Another approach to enhance complex binding to the cell surface
is to use DNAcomplexes of larger size (30). Enhanced binding usually corre-
lates with higher transfection efficiency.
4.2. Crossing the Plasma Membrane and Entering the Cytoplasm
The fact that particle structure of DNAcomplexes is required for DNAdeliv-
ery into cells stimulates the strong notion that endocytosis is the major pathway,
through which DNAmolecules are internalized (48–51). Because the endocy-
totic process ends at the lysosome where DNAis degraded, escape of DNAfrom
the endosome at an early stage of endocytosis is believed to be critical for cy-
tosolic DNAdelivery and is considered to be one of the most important rate-lim-
iting steps that determine overall transfection efficiency. The mechanism of how
DNAmolecules leave the endosome and enter the cytoplasm is unknown. How-
ever, a number of strategies have been explored to enhance endosomal release.
The first of these involves the use of DOPE as a helper lipid for liposome-based
DNAdelivery. It is believed that DOPE is capable of inducing membrane fusion
between the endosome and the liposome through the formation of an inverted
hexagonal structure, which, in turn, results in membrane destabilization and re-
lease of DNAinto the cytoplasm (52). The second strategy is inclusion of mem-
brane fusion proteins or peptides into DNAcomplexes (53). The mechanism of
action of these compounds is similar to that of DOPE: low pH in the early endo-
Chemical Methods for DNADelivery 9
some induces the proteins or peptides to fuse with membrane, releasing the DNA
into the cytoplasm (53). Finally, buffering capacity of charged groups such as the
primary, secondary, and tertiary amines in PEIs and polyamidoamine in den-
drimers may play an important role in endosomal DNArelease in polyplex-me-
diated DNAdelivery. Protonation of these groups in the endosome may create
sufficient osmotic pressure to induce endosomal lysis (35,36).
4.3. DNARelease from Complexes
Although formation of complexes between DNAand synthetic compounds is
essential for cell entry, dissociation of DNAfrom the complexes after the com-
plex has entered the cell appears to be essential for successful transfection. The
necessity for DNArelease from the complexes was demonstrated by Zanber et
al. (54). In their experiments, lipoplexes or free DNAwas injected directly into
the nucleus of oocytes and the level of gene expression in these injected oocytes
was analyzed at a later time. No gene expression was detected in oocytes injected
with lipoplexes as compared to a significant level of gene expression in those in-
jected with free DNA. With respect to cationic liposome-based transfection,
work by Xu and Szoka (55)suggested that DNArelease from the complexes re-
sult from fusion of liposomes and cell membranes. Neutralization of positive
charges of synthetic vector in DNAcomplexes by negatively charged cellular
lipids and other components has been proposed as the mechanism of cytosolic
DNArelease from the complexes (55).
4.4. Nuclear Transport of DNA
Once DNAmolecules are in the cytosol, they must still enter the nucleus. How
this occurs is largely unknown, but the transport of DNAfrom the cytosol into
the nucleus does seem to occur because transgene expression is achieved. Mi-
croinjection of plasmid DNAdirectly into cytoplasm revealed that transport of
DNA from the cytoplasm into the nucleus was of extremely low efficiency
(54,56). An additional factor affecting the transfer of DNAinto the nucleus is nu-
clease activity (56). Cytoplasmic nuclease activity can reduce the amount of in-
tact DNAmolecules available for nuclear import and gene expression, support-
ing the notion that movement of DNAfrom cytoplasm to the nucleus is one of the
most important limitations to a successful gene transfer. Interestingly, studies by
Pollard et al. (57)demonstrated that PEI of 25KDa is able to promote gene de-
livery from the cytoplasm into the nucleus. Such activity, however, is cell type
dependent. Different from cationic liposomes, dissociation of PEI/DNA com-
plexes to allow transcription does not seem to be a problem because injection of
these complexes into the nucleus produces a level of gene product comparable to
injection of plasmid DNAalone (57).
10 Liu et al.
Dean et al. (58)showed that the efficiency of nuclear transport of DNAis de-
pendent on the DNAsequence. Cytosolic injection of DNAcontaining replica-
tion origin and SV40 promoter sequences resulted in a significantly higher level
of DNAimportation into the nucleus than that observed with plasmids without
these specific sequences (58). Inclusion of a nuclear localization signal, nor-
mally a short lysine- and arginine-rich peptide, as part of synthetic vector has
been considered as one of the promising strategies to enhance DNAimport into
the nucleus (59).
5. Optimizing Transfection
In general, expression of the genetically coded information in DNAin a par-
ticular type of cell presents its own particular set of problems that must be over-
come to achieve a high level of expression. Unfortunately, selection of synthetic
compounds for a given type of cell is still largely empirical. There is not a set
of hard-and-fast rules to follow. In fact, a particular synthetic compound is al-
most as likely to be the exception as it is to follow any set of rules. Keeping this
caveat in mind, we will make some general comments that we hope will aid
readers in selecting an initial transfection reagent.
Selecting an appropriate synthetic vector for DNAdelivery may require some
homework on the researcher’s part. The first piece of advice is to consult with
the technical service department of commercial resources (Table 1) for an es-
tablished protocol, because many companies have already optimized the ex-
perimental condition for their products in selected cell types. Researchers could
adopt these conditions if the types of cells to be used in their experiments are
the same or similar. This may significantly reduce the effort required to opti-
mize the experimental conditions.
In cases where there is not enough information available to make a selection,
researchers may wish to obtain transfection reagents from a number of com-
mercial sources to identify the one that gives the best results. Many companies
sell kits that contain multiple reagents. Alternatively, if researchers are new to
this type of study or are dealing with new types of cells, they will have to opti-
mize the experimental conditions themselves following the protocols and sug-
gestions described in the following chapters of this section.
As a standard procedure, the most convenient way to do optimization work
is to use a reporter system. The most commonly used reporter genes for this pur-
pose include those that code for luciferase (Luc), green fluorescence protein
(GFP), b-galactosidase (b-gal), chloramphenicol acetyltransferase (CAT), or se-
creted alkaline phosphatase (SEAP). Most reporter gene-containing plasmids
and related reagents for gene-expression analysis are commercially available
from many resources (Table 2). The following is a brief description of the ad-
vantages and disadvantages of the most commonly used reporter assay systems.
Table 1
C
Commercial Sources for Transfection Reagentsa h
e
m
Company Product Composition i
c
a
Amersham-Biosciences CellPhect Transfection Kit CaPO4 or DEAE-Dextran l
M
800-526-3593
e
www.amershambiosciences.com th
o
Bio-Rad Labs CytoFectene Transfection Reagent Cationic lipid d
s
800-424-6723 f
o
www.bio-rad.com r

D
BD Biosciences–CLONTECH CLONfectinΤΜ Cationic lipid N
800-662-2566 CalPhosΤΜ Calcium phosphate A
www.clontech.com D
e
CPG Inc. GeneLimoΤΜTransfection Reagent Plus Polycationic lipids (cid:1)lipid compound li
v
800-362-2740 GeneLimoΤΜTransfection Reagent Super Polycationic lipids (cid:1)lipid compound e
r
y
www.cpg-biotech.com
Gene Therapy Systems GenePORTERΤΜ DOPE (cid:1)Proprietary compounds
888-428-0558 GenePORTERΤΜ2 Proprietary material
www.genetherapysystems.com BoosterExpressΤΜReagent Kit Proprietary material
PGene GripΤΜVector/Transfection Systems GenePORTER (cid:1)plasmid vector
Glen Research Cytofectin GS Cationic lipid
703-437-6191
www.glenres.com
Invitrogen LipofectAMINE 2000TMReagent Polycationic lipid
800-955-6288 LipofectAMINE PLUSΤΜReagent Polycationic lipid (DOSPA:DOPE)
www.invitrogen.com Lipofectin®Reagent Cationic lipid (DOTMA:DOPE)
Transfection Reagent Optimization System LipofectAMINE PLUSΤΜ(cid:1)Lipofectin®
(cid:1)CellFectin®(cid:1)DMRIE
CellFectin®Reagent Cationic lipopolyamine
1
OligofectAMINEΤΜReagent Proprietary 1