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Introduction to Human Gene Therapy

Gene therapy may be defined as the treatment of human disease by the transfer
of genetic material into specific cells of the patient (1). The advances in molec-
ular biology and biotechnology of the last 30 years have greatly enhanced our
understanding of the genetics of pathogenesis and have led to the identification of
numerous disease-causing genes. With the impending completion of the Human
Genome Project, the list of genetic disease targets is likely to grow tremendously.
It is not difficult to envision treatment of genetic diseases such as hemophilia
(2), muscular dystrophy (3–5), or cystic fibrosis (6) through replacement of errant
genes within the affected cells. However, gene therapy approaches are also being
developed for treatments of virtually all forms of cardiovascular diseases (7), neu-
rological diseases (8–10), infectious diseases (11), wound healing (12), and cancer
(13–15). In these instances, the delivered genes may be intended to augment nat-
urally occurring proteins (eg growth factors or cytokines), to alter the expression
of existing genes facilitating a desired cellular or tissue response, or to produce
cytotoxic proteins or prodrug-activating enzymes, for example, to kill tumor cells
in cancer treatment (13,15) or proliferating endothelial cells to inhibit restenosis
following balloon angioplasty (7,16). Finally, it has been shown that expression
of viral genes within certain tissues can result in an efficient immune response,
leading to the development of DNA vaccines (17).

Because of the potential to revolutionize treatment of a broad range of hu-

man diseases, including some of mankind’s most-hated and currently intractable
afflictions, gene therapy has been heavily investigated over the past decade. More
than 17,000 papers containing the keywords “gene therapy” have appeared in
the MEDLINE database since 1990. The first gene therapy clinical trial, for

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severe combined immunodeficiency caused by a defect in the gene encoding the
enzyme adenosine deaminase, was initiated in 1990 (18). However, it was not
until April 2000 that Cavazzana-Calvo and co-workers (19) reported the first
clinical success by treating two infants with

γ c-severe combined immunodefi-

ciency. Also that year, Kay and co-workers (20) reported positive data in an early
hemophilia clinical trial and Khuri and co-workers (21) reported the successful
completion of a phase II clinical trial using a combination of a viral vector and
traditional chemotherapy to treat recurrent squamous cell carcinoma of the head
and neck. Considering that a total of 596 clinical trials have been completed, are
ongoing, or are pending worldwide (22), the small number of successes has been
disappointing.

A variety of factors is impacting the progress of human gene therapy. The

first step is to identify the proper genes and target cells that will elicit the desired
therapeutic effect. It is then necessary to transfer that gene, often specifically to
the target cells only, with the highest possible efficiency. While short-term gene
expression is sufficient for some applications, such as cancer therapies, long-term
expression is needed for treatment of chronic conditions, including most genetic
diseases. Unfortunately, however, maintenance of the expression has been diffi-
cult. Similarly, for many applications it will be critical to regulate the gene ex-
pression, keeping it at the required levels and also controlling when and how the
gene expression is turned on and off. Finally, one must obviously accomplish each
of these tasks in a way that is safe for the patient. Both toxicity/pathogenicity of
the delivery vehicle and the patient’s immune response to the treatment must be
considered. The majority of these factors, with the exception of identifying a ther-
apeutic gene, are primarily functions of gene delivery. Thus, the key limitation
to development of human gene therapy remains the lack of a safe, efficient, and
controllable methodology for gene delivery (23).

Methods of Gene Delivery

Gene delivery vehicles can be divided into essentially one of two categories: re-
combinant viruses and synthetic vectors. The majority of synthetic vectors, fur-
thermore, can be divided into polymers (the subject of this review) and liposomes.
Each type of material has important advantages and disadvantages. In order to
best understand the problems facing polymer-based gene delivery vehicles and
their current state of development, it is useful to briefly examine the alternate
methodologies (Table 1).

Viral Vectors.

The primary function of a virus is to efficiently carry its own

genome from one host cell to another through (perhaps) “hostile” environments,
enter the new target cell, navigate to the cell nucleus, and initiate expression of its
genome—albeit for the purpose of self-replication. Viruses can be transformed into
gene delivery vehicles by removing part of the virus’ genome and replacing it with
a therapeutic gene. If done carefully, the resulting recombinant virus will retain
the functions essential for infection of specific target cells, but will be rendered in-
capable of replication and, hence, will be nonpathogenic. Because viruses evolved
essentially as sophisticated gene delivery vehicles, such recombinant viral vectors
are incredibly efficient. Viral vectors have been employed in the majority of gene

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Table 1. Comparison of Three Classes of Gene Delivery Vectors

Advantages

Disadvantages

Recombinant

Highly efficient

Lack of cell specificity

viruses

Can infect dividing cells

Pathogenicity

Prolonged expression possible

Immunogenicity

Used in most clinical trials to date

Difficult and expensive

production and purification

Liposomes

Relatively efficient

Poorly stable

Perform well in vivo

Toxicity

Little immunogenicity

Lack of cell-specific targeting

Used in some clinical trials

Polyplexes

Simple

Low efficiency

Inexpensive

Work poorly in vivo

Potential for cell-specific targeting

Not used in clinical trials

Flexible chemistry

to date

Potentially biocompatible

delivery studies reported in the literature and in nearly three quarters of gene
therapy clinical trials (429 of 596 protocols approved as of September 2001) (22).

Viral gene delivery vectors have been based upon retrovirus, lentivirus

(eg, HIV), adenovirus (AdV), adeno-associated virus (AAV), herpes simplex virus
(HSV), and pox virus. There are several important properties that determine the
suitability of a viral vector for a specific indication. The duration and regulation
of gene expression are often critical factors. For example, retrovirus, lentivirus,
and AAV integrate their genetic payload with the host genome, leading to the
potential for prolonged transgene expression. The range of host cells that may be
infected is also very important. Retrovirus, AdV, and AAV can infect a wide vari-
ety of cell types while (wild-type) lentivirus and HSV are limited to specific white
blood cells and neurons, respectively. Furthermore, most retroviruses can infect
only actively dividing cells, which greatly limits the host cell range, especially for
in vivo gene transfer. In contrast, the closely related lentivirus, as well as AdV
and AAV, can infect nondividing cells. For more information on recombinant vi-
ral gene delivery vectors, several excellent reviews have been published recently
(24–28).

While viruses are extremely efficient gene delivery vehicles, they are not

ideal vectors for human gene therapy. Safety concerns have been the primary
bottleneck to clinical application of viral gene delivery vectors. While recombinant
viral vectors have been rendered nonreplicative, and therefore nonpathogenic,
there still exists the possibility for reverting to a wild-type virion or copurifying
replication competent virions. Furthermore, viruses are inherently immunogenic,
not only leading to difficulty with repeat administrations, but also the possibility

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of dangerous immune reactions. In fact, the death of 18-year-old Jesse Gelsinger in
a gene therapy clinical trial in September 1999 was ultimately caused by a severe
immune response to the adenoviral gene delivery vector employed in the study.
Viral vectors are also limited by their natural target-cell specificity. Some viruses
will infect only a limited number of cell types, and their specificity seldom overlaps
with the needs of human gene therapy. Further, some viruses (including AdV, AAV,
and the pseudotyped retro- and lentiviruses) are quite nonspecific, but for in vivo
applications, it is often necessary to target only one type of cell. Finally, because
the viral vectors are complex biological agents, production and purification are
difficult and expensive.

Synthetic Vectors.

Although viruses are incredibly efficient gene deliv-

ery agents, synthetic vectors provide opportunities for improved safety, greater
flexibility, and more facile manufacturing. In general, synthetic vectors are mate-
rials that bind electrostatically to DNA or RNA, condensing the genetic material
into nanometer-scale complexes (a few tens to several hundred nanometers in
diameter) that protect the genes and allow them to enter cells. Such materials
have included cationic peptides, proteins, polymers, and liposomes. Various syn-
thetic vectors, including (diethylamino)ether (DEAE)-dextran (29) and calcium
phosphate (30), have been used extensively for in vitro gene transfer studies since
the 1960s. However, development of nonviral vectors for in vivo gene delivery, es-
pecially clinical applications, has suffered from problems including toxicity, low
gene transfer efficiency, and in vivo instability.

Cationic Lipids.

Use of cationic lipids for gene delivery was first reported in

1987 (31). Since that time, many different lipid structures have been used. Indeed,
greater than 30 products are currently available commercially for in vitro gene
transfer (eg, Transfectam

®

, LipofectAMINE

®

, Lipofectin

®

, and Lipofectace

®

).

These various lipids differ in terms of the nature of their cationic headgroup, their
hydrophobic tails, and the spacers linking the two. Cationic headgroups display-
ing different numbers of charged groups comprising primary, secondary, tertiary,
and quaternary amines have been reported. The lipid tails can be saturated or
unsaturated and of varying length. Also, cholesterol-based structures have been
reported (eg, DC-Chol (32)). The linker can vary in length and hydrophilicity, and
some have been designed to incorporate degradable linkages to enhance biocom-
patibility. These structural properties affect not only the interactions of the lipids
with the polyanionic genetic material, but also influence the membrane proper-
ties of liposomes formed from them, both of which have been found to have a
strong influence on gene delivery efficiency (33). Furthermore, the cationic lipids
typically require a helper lipid, commonly di-oleoyl phosphatidylethanolamine
(DOPE), that provides the membrane fluidity required to mediate efficient endo-
cytosis and liposome-cell membrane fusion (34).

The variety of lipids and lipid mixtures used in gene delivery has resulted

in steadily improving gene transfer efficiencies. This progress has been docu-
mented in several excellent reviews (35–38). As a result of their relatively high
efficiency, cationic lipids have been the most widely studied of the synthetic vec-
tors. “Lipofection” has been routinely employed in both in vitro and in vivo gene
delivery studies, and in several human gene therapy clinical trials (22). However,
lipid-based gene delivery has several critical limitations including lack of cell-
specific targeting capacity, difficulty in reproducibly fabricating liposomes and

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DNA–liposome complexes, and toxicity, especially upon systemic administration
(39).

Cationic Polymers.

Cationic polymers have been used as gene carriers

since the late 1980s (40). Like the cationic lipids, these polymers, including off-the-
shelf materials and polymers specifically designed for gene delivery, have included
a wide variety of chemistries. DNA-binding moieties include primary, secondary,
tertiary, and quaternary amines, as well as other positively charged groups such
as amidines. The charges may reside in the polymer backbone, in pendant groups,
or in grafted oligomers. The polymers themselves comprise linear, branched, and
dendrimeric structures. Because of this flexibility in polymer chemistries that are
available, it may be possible to provide the multiple functions required for efficient
gene delivery while maintaining biocompatibility, facile manufacturing, and ro-
bust and stable formulation of DNA–polymer complexes (polyplexes). As a result,
cationic polymers have great potential for human gene therapy. However, the poor
gene transfer efficiency achieved thus far has limited clinical application of these
vectors. The specific advantages and disadvantages of several important classes
of cationic polymers is described in more detail below.

Barriers to Gene Delivery.

All gene delivery vectors, whether viral or

synthetic, must perform the same task of escorting genes from solution (eg, in a
vial) to the cell nucleus. In order to complete this task, a vector must navigate a
series of obstacles, both extracellular and intracellular (41). Viruses have evolved
a set of functions to address each challenge. As a result, recombinant viral vectors
are highly efficient gene delivery vehicles. In contrast, synthetic vectors are gen-
erally unsatisfactory because they typically lack one, or several, of the necessary
functions. Therefore, consideration of the important barriers to gene delivery in
the next two sections is important in order to understand the limitations of cur-
rently available cationic polymers and is necessary for the rational design of new
polymers.

Extracellular Barriers

Gene delivery vectors face a first set of barriers in transporting genes from the
test tube to the outer membrane of a target cell. These include in vitro barriers,
such as binding and condensing plasmid DNA and maintaining the complex in
solution, as well as in vivo barriers, including stability and survival in the blood
stream, penetrating the blood vessel wall and surrounding tissue, and specific
binding to the target cells of interest. Several of these barriers are discussed in
this section.

Gene Packaging.

Plasmid DNA is a fragile macromolecule and must be

protected from ubiquitous nucleases found both in vivo and in vitro. Binding of
plasmids by cationic polymers provides the needed protection by sterically block-
ing access of enzymes to the DNA backbone. Further, the physical size of plasmid
DNA (

∼3–5,000,000 Da; radius of gyration of several hundred nanometers) pre-

vents it from easily crossing the membrane of most cells. The vector material must,
therefore, condense the DNA into a smaller, more compact structure. Binding and
condensation of genes by the vector material is a necessary first step to efficient
gene delivery.

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Gene delivery vectors bind to DNA through charge–charge interactions be-

tween the negative phosphates along the DNA backbone and positive charges
displayed on the vector material. The process of condensation is entropically
driven; the free energy decrease resulting from the increased translational en-
tropy of counterions and water molecules dominates the binding energy and
loss of entropy in the DNA and polymer (42). Thus, polyplexes form sponta-
neously upon mixing of cationic polymers with plasmid DNA. The resulting parti-
cles are typically toroidal or spherical structures (43,44) with diameters ranging
from a few tens of to several hundred nanometers. Each polyplex particle typ-
ically comprises several DNA molecules along with many polymer chains. The
structure and morphology of polyplexes appears to be kinetically controlled (45)
and often depends on the order of mixing (polymer to DNA solution or DNA to
polymer solution). Efforts have been made to better understand condensation
(42,45,46), but further improvements in the theoretical understanding of the pro-
cess and better physicochemical characterization of the resulting complexes are
needed.

The structure of the polymer can affect DNA binding and condensation.

Linear, branched, and dendrimeric polymers all bind plasmid DNA with similar
strengths, but complexes containing linear poly-

L

-lysine (PLL) or polyamidoamine

(PAMAM) dendrimers tend to aggregate while branched polyethylenimine (PEI)
and “fractured” PAMAM dendrimers (Fig. 1) do not (47). The aggregation appar-
ently leads to decreased gene transfer efficiency. Not surprisingly, the number
of cationic moieties has a strong effect on the polymer–DNA interaction. Several
groups have reported that a minimum of six to eight charges are required for effi-
cient DNA condensation (48–52). The same groups disagree, however, on the effect
of the oligopeptide sequence. Wadhwa and co-workers reported that addition of a
single tryptophan (Trp) residue into PLL oligomers increased DNA binding (48).
In contrast, Plank and co-workers found that Trp residues had no major effect on
DNA binding and, in fact, decreased the “DNA-compacting potency” of the pep-
tides (50). Additionally, placing the cationic moiety nearer the polymer backbone
and keeping the charges to a minimum separation along the polymer backbone
correlated with increased binding (53).

It is important to note that strong binding and efficient DNA condensation

do not correlate directly with gene delivery efficiency. In fact, in some cases the
opposite is true. This is likely because too tight binding prevents transcription
from those plasmids that do reach the nucleus. Thus, a polymer must balance
sufficient binding strength to protect the plasmid during transit to the nucleus
with the ability to release the plasmid, perhaps due to competitive binding of
genomic DNA or anionic lipids (54), once in the nucleus (52).

Solution Stability.

The solution stability of polyplexes depends on the

polymer structure and on the DNA/polymer charge ratio. Neutral polyplexes
quickly form large aggregates, which are generally ineffective gene delivery
agents. In contrast, positively charged polyplexes, as evidenced by zeta potential
measurements, typically remain in solution. However, new studies have shown
that the solubility and aggregate size of even positive complexes is time-dependent
(45).

The concentration ranges over which polyplexes are soluble and stable are

limited. For example, PLL/DNA complexes precipitate above about 20

µg DNA/mL

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Fig. 1.

Structures of several off-the-shelf gene delivery polymers.

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331

(55). Other polymers show similar limits. The limited solubility imposes a maxi-
mum dose that can be administered in a reasonable injection volume.

Solution stability is further modified by the presence of salts and especially

the protein milieu of the in vivo environment. In the presence of physiological
salt concentrations, many polyplexes aggregate even at concentrations at which
they are stable in water, which further limits the achievable dose and can even
be toxic because of embolization of the particulates in the lung. Adsorption of
serum albumin and other negatively charged proteins causes further aggrega-
tion and can lead to rapid clearance of the polyplexes by phagocytic cells and the
reticuloendothelial system (56). Finally, polyplexes containing PLL, a fifth gener-
ation PAMAM dendrimer, and PEI, as well as some lipoplexes, have been shown
to strongly activate the complement system upon intravenous administration
(57).

Modification of polyplexes with poly(ethylene glycol) (PEG), typically grafted

onto the polymer as a “brush,” can stabilize polyplexes against salt-, protein-,
and complement-induced inactivation (58,59). The increased stability due to PE-
Gylation is presumed to result from steric effects leading to decreased particle–
particle and particle–protein interactions. The effect depends on the PEG molec-
ular weight, the grafting density, and the method of attachment of PEG to
the polymer (58,60). Attachment of other hydrophilic polymers such as N-(2-
hydroxypropyl)methacrylamide (HPMA) (59,61–64), oligosaccharides (59,65,66),
sugars (67,68), and proteins (69) had similar effects.

Cell-Specific Targeting.

The degree of target-cell specificity required for

a given gene therapy varies widely. For some applications, such as hemophilia,
the identity of the transduced cells is of little concern so long as sufficient lev-
els of the therapeutic protein are produced. In other applications, such as cystic
fibrosis, the transgene will act only in specific types of cells; expression in other
cell types is largely benign. But in applications such as cancer therapies, wherein
the goal is to kill the target cells, gene delivery to a very specific set of cells is
required.

Polymers generally do not possess a capacity for cell-specific targeting. A typ-

ical polyplex, exhibiting an excess of positively charged groups over the negative
phosphates, will bind to the negatively charged cell surface through nonspecific
electrostatic interactions (70). However, polymers do provide flexible chemistry
for modifying polyplexes with desired targeting moieties allowing both increased
cell uptake and, often, cell specificity. Many membrane-bound receptor proteins
can be used as targets. Derivatization of amino groups with glycosidic moieties,
including galactose, glucose, mannose, and lactose (71–78), as well as other small
molecules such as folate (79,80), provides selective targeting to cell types dis-
playing the appropriate receptor protein. In two classic examples of protein-
mediated targeting, asialoorosomucoid was attached to polylysine (40,81–83) in
order to target the asialoglycoprotein receptor on hepatocytes, while Wagner and
co-workers developed a method for targeting polyplexes to a variety of cell types,
especially hematopoietic cells, through attachment of the iron-transport protein
transferrin (43,84–87). Finally, certain other proteins may provide targeting to
a more limited range of cells. Examples include epidermal growth factor (EGF)
(88,89), antibodies or antibody fragments (90), and integrin-binding sequences
(91).

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The success of a targeting strategy, and the degree of specificity, will depend

on the conjugation chemistry, the length of spacer between ligand and polyplex,
the ligand–receptor binding strength, and the number of targeting ligands per
polyplex. A variety of common cross-linking chemistries have been used to attach
targeting ligands to the polymers including covalent bonds and biotin-streptavidin
(88). However, care must be taken to ensure that the ligand-receptor interaction is
not disrupted by the conjugation chemistry. Schaffer and co-workers found that for
EGF-biotin/avidin-PLL conjugates, short cross-linkers between the EGF and bi-
otin interfered with EGF/EGF-receptor binding, but a 30 ˚A spacer allowed nearly
unhindered binding and resulted in significantly higher gene transfer efficiency.
There also exists a balance between the specific targeting interactions and non-
specific electrostatic binding to the cell surface. Cell uptake and gene expression
were found to be specific only within a narrow window of polymer/DNA ratios
near electroneutrality (92). Finally, there is typically an optimum in ligand va-
lency, because of saturation of both receptor binding and the cell’s internalization
machinery (92). Thus, efficient cell-specific targeting requires careful optimization
of the various parameters that affect cell–surface binding.

Intracellular Barriers

Upon binding to the surface of a target cell and being internalized, gene delivery
vehicles are challenged with a new set of intracellular obstacles. Several stud-
ies have shown that although

>95% of cells in culture may internalize vectors

rather efficiently (

>100,000 copies per cell (93)), only a small fraction, typically

<50%, express the transgene. Polyplexes are generally internalized by endocy-
tosis, and once in the endocytic pathway, polyplexes may be trafficked to lyso-
somes, acidic vesicles filled with degradative enzymes (94). Gene delivery vectors
must escape this pathway into the cytoplasm. Once in the cytoplasm, the vec-
tors must be transported toward the nucleus and subsequently cross the nuclear
membrane. Finally, at some point during this process, the polymer must release
the genes so that the genes can be transcribed in the nucleus. The process is
illustrated in Figure 2. Each step of this process represents a potential efficiency-
limiting barrier, and a vector needs to provide functionality to overcome each
one.

Endolysosomal Escape.

Polyplexes bind to the surface of cells via non-

specific electrostatic interactions and are internalized via adsorptive pinocyto-
sis. Alternatively, polyplexes derivatized with targeting ligands may bind to
specific cell–surface receptors, in which case they are often internalized by
receptor-mediated endocytosis. In either case, the polyplexes become localized
within endocytic vesicles, which isolate the polyplex from the rest of the cell.
The endocytic pathway represents a hostile environment for polyplexes. The first
vesicle, termed the early endosome, fuses with sorting endosomes from which the
internalized material may be transported back to the membrane and out of the
cell by exocytosis. More generally, however, polyplexes are believed to be traf-
ficked into late endosomes, vesicles that rapidly acidify to pH 5–6 because of
the action of an ATPase proton-pump enzyme in the vesicle membrane. Poly-
plexes may subsequently be trafficked into lysosomes, which further acidify to

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333

Fig. 2.

Intracellular trafficking of polymer gene delivery vectors. 1, Internalization (of-

ten by adsorptive pinocytosis or receptor-mediated endocytosis); 2, escape from endocytic
vesicles; 3, transport through the cytosol; 4, transport across the nuclear membrane. In
addition, polyplexes must be unpackaged at some point in the process, although it is not
known where in the process unpackaging occurs.

pH

∼4.5 and contain various degradative nucleases, proteases, and lipases. It

is believed that polyplexes will become trapped in these vesicles and be de-
graded. Only those vectors that escape into the cytoplasm can go on to reach the
nucleus.

Several strategies have been employed to overcome this barrier. Concur-

rently, treating cells at the time of transfection with chloroquine, which is known
to accumulate in the acidic vesicles and buffer their pH, results in improved gene
delivery (95). While this approach has been commonly employed in in vitro stud-
ies with polymers such as PLL, it is impractical for in vivo gene delivery. Other
researchers have tried to overcome this barrier by incorporating additional func-
tional groups into polyplexes to provide for endosomal escape. For example, cova-
lent attachment of whole, inactivated AdV particles to PLL was found to enhance
gene transfer up to 2000-fold (96–99). It was proposed that this enhancement is
due to more efficient endosomal escape, but the virion particle may also provide
functionality for addressing subsequent barriers (see below). While successful,
this approach is not practical because of the increased difficulty of preparing the
vector and the safety concerns, especially immunogenicity, raised by the presence
of the virus. Alternatively, fusogenic viral (100–103) or synthetic (104–108) pep-
tides may also provide the endosomal escape function (109,110). These peptides
are typically pH-sensitive amphiphiles that undergo a structural change, such as
α-helix formation, at acidic pH and then insert into and disrupt the vesicle mem-
brane. The peptides may be associated with polyplexes via electrostatic interac-
tions or covalently attached to the polymer. Gene transfer is typically enhanced
1–3 orders of magnitude in this fashion.

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Because of the versatile structures available in cationic polymer gene de-

livery vehicles, it is possible to build an endosomal escape function into the
polymer itself. Polymers such as PEI and PAMAM dendrimers, which contain
a large number of secondary and tertiary amines, can undergo large changes in
protonation between physiological pH and lysosomal pH. It has been hypothe-
sized that these polymers can, in fact, prevent acidification of endocytic vesicles.
Furthermore, these polymers may act as “proton sponges” wherein their buffer-
ing capacity results in osmotic swelling and rupture of the endocytic vesicles
(111–113). While it is not clear how these polymers function (whether simply
by protecting the DNA from acidic pH and enzymatic degradation or by osmotic
swelling of the vesicles), there is a clear relationship between polymer buffer-
ing capacity and gene delivery (47). Indeed, polymers designed specifically to
provide such buffering capacity have proven to be highly successful (vide infra)
(114–117).

Transport through the Cytoplasm.

Once released from endosomal com-

partments, polyplexes must move through the cytoplasm to the nucleus. How-
ever, the cytoplasm is quite concentrated with protein, microtubules, and other
organelles, all of which may hinder polyplex transport. In fact, previous studies
have indicated that movement through the cytoplasm is size-dependent. Fluores-
cence recovery after photobleaching studies showed that dextran polymers with
hydrodynamic radius more than 260 ˚A appear to be immobile (118). More recent
studies of DNA mobility showed again that diffusion is size-dependent, with DNA
larger than 3000 bp being essentially immobile (119). Given the large size of poly-
plexes, one might expect these vectors to be immobile as well. This fact, together
with the known degradation of DNA in the cytosol as a result of the presence of
cytosolic nucleases (120), presents an often-overlooked barrier to efficient gene
delivery.

Viruses, in contrast, typically have evolved mechanisms for cytoplasmic

transport. Subgroup C adenoviruses, including types 2 and 5 (Ad2 and Ad5) es-
cape endosomes rapidly following endocytosis and, thus, are typically “far” from
the nucleus. These virus particles have evolved mechanisms by which they then
use cytoplasmic dynein motor proteins for active transport along microtubules
(MT) toward the nucleus. In contrast, subgroup B Ad (eg, Ad7) lacks the abil-
ity to utilize dynein-mediated MT transport. Rather, Ad7 remains in endocytic
vesicles and is transported toward the nucleus with their endocytic vesicles (as
early endosomes mature into late endosomes and finally lysosomes, the vesicles
are transported along MT toward the nuclear envelope). Ad7 then escapes from
lysosomes (triggered by lower pH) near the nucleus, thereby mitigating the ef-
fects of low cytoplasmic motility (121). It may be possible that positively charged
polyplexes use similar mechanisms by nonspecifically interacting with motor
proteins, for example. Alternatively, polyplexes may be redistributed through-
out the cell, with some ending up near the nucleus, becasue of the mixing that
occurs during mitosis. The mechanisms of cytoplasmic polyplex transport need
to be better characterized in order to facilitate design of improved polymeric
vectors.

Nuclear Localization.

Therapeutic genes must reach the cell nucleus to

be expressed by the target cell. However, because the genome and nuclear ma-
chinery are vital to a cell’s functions, nature has isolated the nucleus behind a

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335

double-bilayer membrane with tightly regulated pores to allow import and export
of a very specific set of biomolecules. The nuclear pore complex, a 10

7

-kDa assem-

bly of at least 30 distinct proteins, allows passage of small molecules, but proteins
larger than 10–20 kDa require active transport via specific nuclear import pro-
teins (eg, importins). Viruses have evolved mechanisms to utilize the cell’s nuclear
import machinery; polymers clearly do not have this capacity. Transfection imme-
diately before cell division (and concomitant disruption of the nuclear membrane)
is 30- to 500-fold more effective than transfection of cells at the beginning of their
cell cycle (122). In contrast, nondividing cells are poorly transfected compared to
dividing cells (122,124). Thus, transport across the nuclear membrane, especially
for nondividing cells, appears to be a formidable obstacle to nonviral gene delivery
vectors.

It may be possible to modify polyplex vectors to allow efficient nuclear im-

port (125). For example, it is well known that many proteins are targeted to the
nucleus by the presence of nuclear localization signals (NLS), short peptide se-
quences, typically highly cationic, that are recognized by importins. Because of
their positive surface charge, it is possible that polyplexes could mimic NLS to a
limited extent (126), but if so they are very inefficient as very few vectors typically
reach the nucleus (127). However, there is some evidence that this is not a general
phenomenon. Other polymers and cationic liposomes do not show an NLS effect
(128,129). Several investigators have modified polyplexes by attachment of NLS
with generally modest results (128–132). Further, nucleotide sequences on the
genes themselves may provide some nuclear targeting (133–135), but this will not
find general use as the nucleotide sequences are hidden by the polymer in most
cases. In short, nuclear import of polyplex vectors is one of the most poorly char-
acterized steps in the gene delivery process. A better understanding of nuclear
transport mechanisms is needed.

Unpackaging.

Just as incorporation into a polymer complex protects DNA

from enzymatic degradation, so too will complexation prevent binding of the
proteins required for initiation of gene expression. Thus, a gene delivery vec-
tor must release its DNA at some point in the delivery process. Indeed, effi-
cient nuclear localization does not correlate with high gene expression. Sev-
eral studies have found that reducing the polymer/DNA binding strength, by
reducing the number of positive charges (136), conjugation of PEG chains (60),
or decreasing the polymer molecular weight (52), leads to increased gene ex-
pression. Polymers must clearly be designed to incorporate a mechanism for
nonspecific or environmentally responsive release of the genes, ideally in the
nucleus.

“Off-the-Shelf” Polymers in Gene Delivery

Many of the early studies on polymer-mediated gene delivery employed commer-
cially available polymers. As they were not designed for gene delivery, their effi-
cacy as delivery vectors is somewhat serendipitous. Off-the-shelf polymers have
nonetheless been very widely studied and form the basis for much of the gene de-
livery polymer literature. However, significant problems face these polymers, most

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GENE-DELIVERY POLYMERS

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notably with cytotoxicity. Three of the major off-the-shelf polymers are described
below.

Poly-

L

-lysine.

Poly-

L

-lysine (PLL; Fig. 1) was one of the first cationic poly-

mers used in the modern era of gene delivery research (40,55). It is commercially
available in a range of sizes from oligomers to several hundred kilodaltons. For
gene delivery, 20–50 kDa PLL is most commonly used. Polyplexes of DNA and
PLL itself are poor gene delivery vectors, but addition of targeting ligands greatly
enhances in vitro and in vivo gene delivery efficiency.

Some of the first reports of PLL-mediated gene delivery employed conjuga-

tion of the asialoorosomucoid glycoprotein to target the asialoglycoprotein receptor
on mouse hepatocytes in vitro and in vivo (40,81,82). Shortly thereafter, a series of
papers by Wagner and colleagues developed a process, termed transferrinfection,
based on conjugation of the iron transport protein, transferrin, to PLL (reviewed
in Refs. 87 and 137). These thorough studies provided a firm basis for much of the
subsequent gene delivery literature, including some of the first studies of polyplex
formation (43) and mechanisms of endolysosomal escape (86,99,100). PLL/DNA
complexes have also been targeted to specific cells through conjugation of sugars
(49,71–73,136,138), folate (79), RGD-displaying peptides (91,139), and antibodies
(140).

Early studies on PLL were promising, but it now appears unlikely that PLL-

based polyplexes will reach clinical application. Development has been limited by
the relatively low efficiency. This is generally accepted to be the result of poor
escape from the endocytic pathway. In recent years, PLL has been relegated to a
role in basic mechanistic studies or as a point of comparison in studies of more
promising polymers.

Polyethylenimine.

PEI has been used as a gene delivery vector since 1995

(111). The polymer has been known for many years and has traditionally been
used as a chelator, in water purification, and in shampoos. It is available in two
main forms: linear and branched (Fig. 1). The branched polymer is synthesized via
acid-catalyzed polymerization of aziridine, while the linear form is synthesized in
a similar way but at reduced reaction temperatures (141). A range of molecular
weights (eg,

<1, 2, 22, 25, 50, 70, and 800 kDa) are commercially available from

several manufacturers.

PEI is one of the most effective gene delivery polymers studied to date (142).

Depending on the cell line, it is typically several orders of magnitude more efficient
than PLL. Importantly, PEI mediates gene delivery efficiently in the absence of
any exogenous endosomolytic agent such as chloroquine, inactivated viruses, or
fusogenic peptides. PEI-containing polyplexes have been targeted to specific cell
types by the conjugation of ligands including galactose (74,76), mannose (143),
transferrin (90,144), and antibodies (145). Also, PEI has been highly successful
for in vivo gene delivery to a variety of tissues including the central nervous
system (146,147), kidney (148), lung (149,150), and tumor (151) (reviewed in
Ref. 142).

The relatively high gene transfer efficiency mediated by PEI is believed to be

due in large part to efficient escape from the endocytic pathway through a proposed
mechanism known as the “proton-sponge” hypothesis (111,112) (Fig. 3). Endocytic
vesicles are acidified by the action of an ATPase enzyme that actively transports
protons from the cytosol into the vesicle. Because every third atom of the polymer

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GENE-DELIVERY POLYMERS

337

Fig. 3.

Schematic of endocytic vesicle swelling and rupture as proposed by the “proton-

sponge” hypothesis. The polymer, containing unprotonated basic moieties at neutral pH,
buffers the pH within the vesicle and causes the ATPase to pump excess protons into the
compartment. The concomitant influx of counterions increases the osmotic pressure across
the membrane, causing the vesicle to swell and ultimately rupture.

is a nitrogen, PEI has a very high density of protonatable amines, only 15–20% of
which are protonated at physiological pH (152). As a result, PEI will tend to buffer
the endosomes causing the ATPase to transport many more protons to reach the
desired pH. The accumulation of protons in the vesicle must be balanced by an
influx of counterions, typically chloride. The increased ion concentration results
in osmotic swelling of the vesicle and, eventually, in the rupture of the endosome
membrane.

Despite its advantages, development of PEI gene delivery systems has been

hindered by the polymer’s relatively high cytotoxicity. Toxicity appears to be de-
pendent on the polymer molecular weight. Recently, it has been shown that PEI as
small as 10 kDa is a relatively successful vector with very low toxicity (153,154).
Based on these findings, Gosselin and co-workers (155) generated a novel vec-
tor by cross-linking 800 Da PEI with disulfide linker groups. They hypothesized
that the cross-links would be cleaved upon exposure to the reducing environment
of the cytoplasm, presumably leading to lower toxicity. Gene expression medi-
ated by these polymers was slightly reduced relative to 25 kDa PEI, but for some
of the polymers cytotoxicity was virtually eliminated. Taking a different tack,
Han and co-workers (156) synthesized a cholesterol-derivatized, low-molecular-
weight PEI (termed water-soluble lipopolymer) that is less toxic than the stan-
dard 25-kDa PEI and mediates two- to threefold higher gene delivery efficiency, as
well.

Polyamidoamine

Dendrimers.

Polyamidoamine

(“Starburst”)

den-

drimers are spheroidal, cascade polymers (Fig. 1) that can be synthesized from
an ammonia or ethylenediamine core by successive addition of methyl acrylate
and ethylenediamine (157). The size of the polymer, and importantly the surface
charge, are controlled by varying the number of “generations” in the synthesis.
Haensler and Szoka (113) originally reported the use of PAMAM dendrimers for
gene delivery. They examined dendrimers of generations 1–10 and found that
the sixth generation dendrimer was better than the others by

∼10-fold. Because

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GENE-DELIVERY POLYMERS

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of the large number of secondary and tertiary amines on the polymer, and its
resulting buffering capacity, PAMAM dendrimers are thought to efficiently escape
from the endocytic trafficking pathway by a mechanism similar to PEI. Indeed,
gene delivery is unaffected by chloroquine, suggesting that endosomal escape
is not limiting. Because of its relatively high gene delivery efficiency and good
biocompatibility, PAMAM dendrimers have recently been employed in several in
vivo gene delivery studies (158–161).

Interestingly, a partially degraded form of PAMAM dendrimer appears to be

a more effective gene transfer agent than the intact polymer. Heat treatment of the
polymer in various solvolytic solvents (eg, water or butanol) significantly degrades
the polymer at the amide linkages, resulting in a heterodisperse population of
compounds with molecular weight ranging from

<1500 kDa to tens of kilodaltons.

These “fractured” dendrimers show

>50-fold enhanced transfection activity (162).

The mechanism of the enhancement appears to be twofold. First, the fractured
dendrimer exhibits greater flexibility, allowing a more beneficial interaction with
the plasmid DNA (162). Second, polyplexes containing fractured PAMAM appear
to have enhanced solution stability in comparison to polyplexes containing intact
polymer, which tend to aggregate (47).

Polymers Designed for Gene Delivery

Many different types of polymers have been specifically designed for use as gene
delivery vectors. In many cases, the polymers were designed to address one of
the perceived gene delivery barriers; for example, DNA packaging and stabil-
ity in vivo, biocompatibility, and endosomal escape have all been used as design
criteria. The results of such studies have been mixed, with some polymers per-
forming as well as, or even slightly better than, PEI and the PAMAM dendrimers.
In the following, we cannot describe all of the polymers designed for gene deliv-
ery. Rather, we have chosen several new classes of polymers that appear to show
promise.

Imidazole-Containing Polymers.

“Proton-sponge” polymers are some of

the best available off-the-shelf materials for gene delivery. However, they are lim-
ited by considerable cytotoxicity in some cell lines. It would be desirable to mimic
the proton-sponge mechanism, which requires high buffering capacity between
physiological and lysosomal pH, in a polymer that is more biocompatible. Several
groups have recognized that imidazole exhibits the required protonation prop-
erties (pK

a

∼6). Further, because imidazole is a component of several biological

species (eg, the amino acid histidine), polymers incorporating imidazole may be
expected to show increased biocompatibility.

One approach that has been reported was to use a homopolymer of histi-

dine (pHis) (MW

∼11,000) that was derivatized with gluconic acid to increase its

solubility in aqueous solutions (G-pHis) (Fig. 4) (114). The polymer was shown
to efficiently condense plasmid DNA into particles of

∼100 nm diameter. Ternary

complexes of DNA, G-pHis, and transferrin–polylysine conjugates (Tf-PLL) trans-
fected COS-7 cells, in the absence of any exogenous endosomolytic agents, with
efficiency similar to that of DNA/Tf-PLL complexes that required addition of

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GENE-DELIVERY POLYMERS

339

Fig. 4.

Imidazole-containing, biocompatible “proton-sponge” polymers (114,115,117).

chloroquine. Further, the polymer showed negligible toxicity at concentrations
employed in gene delivery studies.

A second approach is to derivatize PLL with imidazole-containing pendant

groups (Fig. 4). Three chemistries have been reported. In the first, the ¯

ε-amino

groups of PLL or oligolysine were derivatized with (Boc-protected) histidine, re-
sulting in an amide linkage with a primary amine (from the histidine) remaining
(117,163). Thus, the imidazole group was introduced without reduction of the
number of positive charges available for binding DNA. In the second, PLL was
derivatized with imidazoleacetic acid yielding a similar amide linkage, but in the
absence of the primary amine (115). Both polymers were found to efficiently trans-
fect a variety of cell types in the absence of any exogenous endosomolytic agent,
and gene expression levels increased with increasing imidazole content. Gene ex-
pression levels mediated by histidylated PLL were 3–4 orders of magnitude higher
than with underivatized PLL (117). In fact, Putnam and co-workers reported gene
expression levels comparable to those achieved with PEI polyplexes, yet in the ab-
sence of any cytotoxicity (under the same conditions, PEI reduced cell viability by
∼80%). Finally, Benns and co-workers (164) reported a comb-type polymer of poly-
histidine grafted onto the

ε-amino groups of polylysine. This polymer was slight

more efficient than PLL, but was still enhanced approximately two-fold by the
addition of chloroquine.

Cyclodextrin-Containing Polymers.

Cyclodextrins (CD) are cyclic

oligomers of ¯

α-1,4-linked glucopyranose units that exhibit interesting guest–host

properties. The cavity of the CD ring is relatively hydrophobic. Thus, CDs form
inclusion complexes with many small hydrophobic molecules. This property has

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GENE-DELIVERY POLYMERS

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Fig. 5.

Example of a cyclodextrin-containing polymer incorporating amidine linkages

(166).

been exploited for drug delivery applications. An excellent review of CDs is avail-
able (165).

Linear CD-containing polymers for gene delivery have been synthesized by

condensation of diamino-CD monomers with diimidate comonomers to yield a
diamidine-containing polycation (Fig. 5) (166,167). The CDs were functionalized
with amino groups of varying structure placed on opposite sides of the CD ring
(166). Further, the length and chemistry of the spacer between the amidine moi-
eties was systematically varied from 4 to 10 methylene units (167). Typically, the
syntheses yielded highly water-soluble oligomers with degree of polymerization
from 4 to 6 and molecular weights between 5800 and 8800. These polymers con-
densed plasmid DNA into particles of 100–150 nm in diameter at

+/− charge ratios

greater than 5, as measured by dynamic light scattering and transmission electron
microscopy, and transfect cells at

+/− ratios greater than 10. The best polymers

of this class transfected cells in culture with efficiencies comparable to PEI and
Lipofectamine. Transfection was enhanced 20-fold by the addition of chloroquine
suggesting that, as for many other polymers, escape from the endocytic pathway
is an efficiency-limiting barrier.

An important aspect of these polymers is their lack of cytotoxicity. The IC

50

(polymer concentration at which cell viability is reduced by 50%) is in the millimo-
lar range. Furthermore, the LD

40

(dose of polymer in milligrams per kilogram that

will cause death in 40% of the animals) in mice for the polymer shown in Figure 5
was found to be 200 mg/kg. These values are at least 10-fold higher than for other
gene delivery polymers such as PEI. The low toxicity is clearly due to the presence
of the CD (167).

Finally, it is interesting to note that the CD moieties provide an interesting

opportunity for modification of the polyplexes with a variety of functionalities. For
example, it may be possible to introduce receptor-targeting or nuclear-localization
moieties with a bifunctional molecule containing the targeting moiety on one end
and a CD-intercalating moiety (eg, adamantane) on the other end. Such a rela-
tively simple route to introduce structural and functional diversity to the poly-
plexes may have many exciting applications.

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GENE-DELIVERY POLYMERS

341

Degradable Polycations.

Two of the important barriers to polymeric

gene delivery, unpackaging of the plasmid and cytotoxicity, have recently been ad-
dressed via synthesis of biodegradable polycations. For example, several cationic
polyesters have been reported (168–171). One of these polymers, poly[ ¯

α-(4-

aminobutyl)-

L

-glycolic acid] (PAGA) was designed as a biodegradable mimic of

PLL (170). PAGA (MW

∼3300) degraded quickly in aqueous solution at pH 7.3

(37

◦

C), but the degradation process was much slower for the polymer in polyplexes

with plasmid DNA. PAGA showed no cytotoxicity under conditions where PLL
reduced cell viability by 80%. In the presence of chloroquine, PAGA transfected
cells threefold better than PLL at optimized conditions (170). Furthermore, PAGA
has shown efficacy in delivering plasmid DNA in vivo by reducing the severity of
autoimmune insulitis in nonobese diabetic mice (172,173) and in cytokine gene
delivery for cancer treatment (174). Similarly, a hyperbranched poly(amino es-
ter) was synthesized as a biodegradable mimic of PEI and/or PAMAM dendrimers
(171). This polymer was minimally toxic in comparison to PEI and PAMAM den-
drimers. Transfection efficiency was 1–2 orders of magnitude less efficient than
PEI or PAMAM, but was 10-fold better than PAGA (without chloroquine).

Another class of polymers that has recently been developed for gene delivery

are the poly( ¯

β-amino esters), synthesized by the addition of primary or secondary

amines to diacrylates (175–177). Initially, a set of three poly(

β-amino esters)

(Fig. 6) was shown to be capable of binding and condensing plasmid DNA and
exhibited negligible cytotoxicity to cells in culture (175). Subsequently, the au-
thors employed a combinatorial approach to synthesis of a library of poly(

β-amino

esters) (177). Seven diacrylate monomers were reacted with 20 amine monomers
to yield 140 structurally unique polymers (MW 2,000–50,000). The library was
screened for solubility in aqueous buffer (pH 5) and DNA binding by gel elec-
trophoresis. Fifty-six of the 70 water-soluble polymers interacted sufficiently with
DNA to allow transfection studies. Six polymers were identified as “hits” worthy
of further study, and two of the polymers mediated gene delivery similar to or
exceeding that observed with PEI.

Another novel approach has been to employ disulfide-containing moieties

that will be cleaved in a reducing medium such as the cytosol and nucleus. In one

Fig. 6.

Biodegradable poly(

β-amino ester) polycations synthesized from the addition of

diamine monomers to diacrylate comonomers (175).

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GENE-DELIVERY POLYMERS

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such polymer, poly[Lys-(AEDTP)], the lysine

ε-amino groups are substituted with

3-(2-aminoethyldithio)-propionyl residues such that the amino groups interacting
with the DNA phosphates are linked to the polymer via a disulfide bond (178).
Decondensation of polyplexes was observed in the presence of reducing agents
including dithiothreitol and glutathione. Transfection of HepG2 cells was 50-fold
more efficient with poly[Lys-(AEDTP)] compared to PLL polyplexes. PEI (800 Da)
was reacted with two cross-linking reagents, dithiobis(succinimidylpropionate)
and dimethyl-3,3

-dithiobispropionimidate, at two different amine cross-link ra-

tios to generate a set of reducible conjugates (155). The gene transfer efficiency
was found to be proportional to conjugate size. While none of the polymers was as
effective as the commonly used 25-kDa PEI, they were all less toxic.

Conclusions

A variety of polymers has been employed in gene delivery studies to date, but
their effectiveness as gene therapy vectors remains orders of magnitude poorer
than viral vectors. As a result, polymers are generally considered unacceptable for
clinical applications. The important extra- and intracellular barriers to efficient
gene delivery are known. The lack of efficiency of polymer gene delivery vectors is
due to a lack of functionality for overcoming one or several of these barriers. Based
on the large number of studies of off-the-shelf gene delivery polymers, much has
been learned about the structure–function relationships of polymer vectors. This
knowledge has been applied to design and synthesis of new polymers, tailor-made
for gene delivery, and a number of promising candidates have been reported in
recent years. With our growing understanding of polymer gene delivery mecha-
nisms and the continued efforts of creative and talented polymer chemists, it is
likely that polymer-based gene delivery systems will become an important tool for
human gene therapy.

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D

ANIEL

W. P

ACK

University of Illinois at Urbana-Champaign

GLASS TRANSITION.

See Volume 2.