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INTRODUCTION |
Receptor-mediated gene transfer has great appeal as a strategy for
gene therapy because of its specificity and low toxicity in
vivo but has drawbacks, including low level and transient gene expression (1-9). Targeting the serpin (serine
protease inhibitor) enzyme complex receptor
(SEC-R)1 might transfect many
cell types that are potentially interesting for gene therapy, including
hepatocytes, macrophages, and neurons (10-14). Ligand-conjugated
poly-L-lysine (poly K)-DNA complexes directed at this
receptor deliver reporter genes, specifically, to receptor-bearing
cells. The synthetic ligands are based in sequence on amino acids
346-374 of human
1-antitrypsin (9). We undertook a
systematic study of the contribution of the protein portion of the
complex to intensity and duration of gene expression.
Prior studies of the composition of the protein portion of
receptor-directed gene transfer complexes have focused on the degree of
substitution (6, 15). One study did not find a consistent relationship
between chain length and gene expression in vitro (6). No
study has examined the relation between the chain length of poly K used
to condense DNA and duration of expression. Moreover, it is not clear
whether modifications worked out in an in vitro model can be
applied to the in vivo situation (16, 17).
Our system has several advantages for studies of this type, including
the ability to determine the extent of lysine substitution by linker
and ligand, even at very low levels, with NMR. Furthermore, the same
ligands bind to the receptors of human, mouse, and rat, thus allowing
parallel examination in vitro in human cells and in
vivo in animal models. We used this system to investigate the influence of chain length of poly K used on the intensity and duration
of gene expression in vitro and in vivo. Using
our in vitro model, we compared the optimally substituted
poly K molecules of average chain length 36 with those of average chain
length 256. Longer chain poly K molecules gave significantly longer
duration of expression both in vitro and in vivo
than shorter chain poly K molecules for a given ligand and degree of substitution.
Manipulation of the protein portion of receptor-targeted DNA complexes
strongly influenced the duration and intensity of gene expression. This
demonstration, in vitro and in vivo, addresses some of the limitations of the system and provides a new basis for
improvement of this molecular conjugate for therapeutic purposes.
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EXPERIMENTAL PROCEDURES |
Materials--
DNA-modifying enzymes, nucleotides, and
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside were
purchased from Boehringer Mannheim.
-Galactosidase luminescent
activity was measured using reagents obtained from Tropix (Bedford,
MA). Poly K was obtained from Sigma, and LC
sulfo-N-succinimidyl-3-(2-pyridyldithio) proprionate
(sulfo-LC SPDP) was purchased from Pierce. Luciferase activity was
measured using Promega (Madison, WI) assay reagents. Sephadex G-10
columns and Bradford protein assay reagents were obtained from Bio-Rad. Peptides C105Y (CS IPPEV KFNKP FVYLI) and C1315 (CFLE AIPMS IPPEV KFNKP FVFLI IHRD) were synthesized by solid phase method, purified, and subjected to amino acid composition and sequence
analysis as described previously (10). The 5-amino acid binding motif,
FVFLI, shown in bold type, is sufficient for SEC-R binding (12).
Cell Culture--
HuH7 cells were maintained as described
previously (9).
Construction of the Protein Portion of DNA
Complexes--
Peptides C1315 and C105Y were covalently linked to
different size poly Ks (average poly K length, 36 amino acids (9.7 kDa) and 256 amino acids (53.7 kDa)) using the heterobifunctional
cross-linking reagent sulfo-LC SPDP as described previously (18). To
account for the differences in the size of the poly Ks, reaction
concentrations were normalized to moles of lysine residues present in
each poly K. Aliquots of each of the poly Ks were mixed with sulfo-LC
SPDP in 0.1 M phosphate-buffered saline, pH 7.4, at room
temperature (about 22 °C) for 30 min. The reaction mixtures were
then dialyzed exhaustively to remove unreacted sulfo-LC SPDP and low
molecular mass reaction products, and samples were reserved for NMR
analysis. Poly K linker conjugates were then mixed with C1315 or C105Y
at 22 °C for 24 h. Aliquots were set aside for NMR analysis.
Two approaches were taken to conjugate construction. In one, all the
poly K used to condense DNA was substituted randomly with sulfo-LC SPDP
and then with ligand. These are termed "random substitution"
experiments and produced poly Ks substituted on either 3.5 or 7.8% of
lysines with sulfo-LC SPDP. Each of these constructs was then
substituted with ligand on 0.017, 0.097, or 0.27% of the lysine
residues. These values were determined by NMR. When these substituted
poly K molecules were complexed to charge neutrality with the pGL3
plasmid, the complexes contained ~2, ~10, or ~35 ligands.
The second approach was to adjust conditions to substitute only one
lysine on each poly K molecule with sulfo-LC SPDP-ligand. Substituted
poly K molecules were mixed with unsubstituted poly K of the same chain
length to give mixtures that, when complexed to charge neutrality to
the pGL3 plasmid, had a specified number of ligands/DNA molecule,
depending on the relative proportions of substituted and unsubstituted
poly K in the mixture. This approach was termed the "ligand
dilution" method. Optimal ligand to DNA ratios were then used to
construct CMV lacZ II complexes for animal experiments.
NMR Spectra--
To verify the rates of substitution of the
different poly Ks as well as examine the structure of each of the
peptides, we used NMR. An aliquot (5-10 mg) of the conjugate was
exhaustively dialyzed against water, lyophilized from water and
subsequently from D2O, and then resuspended in 0.75 ml of
99.99% D2O (Aldrich) or 90%
Me2SO-d6, 10% D2O.
Proton NMR spectra were obtained at 600 MHz on a Varian Unity Plus 600 NMR spectrometer using standard proton parameter, acquired over 0.5-16
h. Chemical shifts were referenced to the residual HDO resonance at 4.8 ppm or the Me2SO-d6 multiplet at 2.5 ppm. Spectra of C1315 peptide and C1315-poly K conjugate were obtained
in 90% Me2SO-d6. Aliquots of
dialysis bath water as well as dialysis bag wash were also lyophilized and examined by NMR to verify the absence of contaminants. There was
close agreement in calculated efficiency among reactions containing similar ratios of sulfo-LC SPDP and poly K, expressed as lysine residues available.
Reporter Genes and Plasmid Preparation--
Two plasmids coding
for two different reporter genes were used. The expression plasmid,
pGL3 control (size, 5.3 kb; Promega) contained the SV40 viral promoter
and enhancer ligated to the Photinus pyralis luciferase gene
and inserted into the Escherichia coli pUC19 vector. The
plasmids pCMV lacZ II (size, 10.8 kb; gift from Dr. Lloyd
Culp, Case Western Reserve University) (19) contained the CMV promoter
ligated to the E. coli
-galactosidase (lacZ) gene. Plasmids were purified twice on a CsCl density gradient (20).
Identity of the plasmids was confirmed by restriction endonuclease
digestion, and purity was established by 1.0% agarose gel electrophoresis.
Preparation of the C1315 and C105Y Peptide-based DNA
Complexes--
The carrier-DNA complexes were prepared using general
techniques previously described (5). Plasmid DNA was condensed by slow
addition (5 µl over 5 min) of the C1315 and C105Y-poly K conjugates
in 400 mM NaCl under constant vortexing at room
temperature. Sufficient carrier was added so that the charge on poly K
neutralized the negative charge on the phosphate groups of DNA. With
addition of the carrier to the DNA, aggregates appeared, and the salt
concentration was adjusted by small aliquots of 5 M NaCl.
As the ionic strength rose, the aggregated DNA complexes assumed a
condensed state, and the turbidity of the solution cleared (5). The
final volume of the solutions was typically 500 µl (0.5-1 µg of
plasmid DNA/5 µl), containing a mixture of 1:0.45 w/w DNA to
peptide/poly K conjugate ratio in 0.8-1 M NaCl. For
controls, DNA was condensed in the same method with unconjugated poly
K. Final concentrations of NaCl differed according to the affinities of
the different conjugates for DNA (5). Aliquots of each reaction mixture
were examined by electron microscopy to assess condensation.
Electron Microscopy of the Condensed DNA
Complexes--
Micrograph grids were prepared as described previously
(3, 5). Briefly, immediately after formation of DNA complexes, a drop
of a solution (1:10 dilution of complex mixture in water) was added to
a 1000 mesh electron microscope carbon grid, blotted, and stained with
0.04% uranyl acetate. After rotary shadowing, samples were examined
using a JEOL-100C electron microscope.
Atomic Force Microscopy (AFM)--
Samples were prepared on
suitable substrate surfaces and scanned by atomic force microscopy as
described previously (21, 22). Briefly, immediately after formation of
DNA complexes, a drop of the solution (1:100 dilution) was applied the
surface of a 2 × 2-cm Mica wafer, dried for 3 h, and scanned
by a Nanoscope III atomic force microscope. The cantilever deflection
maintains an applied force less than 10 nN; the force of
adhesion is about 30 nN. Feedback gain and scanning speed
are adjusted to minimize errors due to temporal response limitations.
Samples scanned in solution were immersed in water for 1 h in a
sealed chamber, and scanned. Images were transferred in binary format
to a SPARC 10 Sun Microsystem workstation, converted to gray scale, and analyzed.
Transfection of Cells in Culture--
HuH7 cells were
transfected as described previously (9). Controls included:
(a) HuH7 cells transfected with 1.2 pmol of pGL3 control
condensed with unconjugated poly K in the presence of free C1315 or
C105Y peptides and free sulfo-LC SPDP linker (comparable with peptide
and linker content in the conjugated complexes); (b) HuH7
cells transfected with 1.0 pmol of pGL3 control DNA by Lipofectin; and
(c) HuH7 cells transfected with 1.11 pmol of unconjugated
poly K condensed DNA by Lipofectin. Control (a) was designed
to test for nonspecific uptake; controls (b) and (c) confirmed that target cells could express the transgene.
After addition of the complex and/or Lipofectin, all cells were
incubated at 37 °C for 3 h. Cells were then rinsed with
Ca2+/Mg2+ phosphate-buffered saline, and fresh
growth medium was added and incubated at 37 °C (with a change of
medium every 2 days) until the functional assay was performed. All
transfections were done in duplicate. No excess cell death was observed
in any of the wells transfected with the DNA-conjugated poly K
complexes throughout the incubation. Luciferase expression was measured at 2, 4, 10, and 16 days after transfection. Number of samples (N) denotes transfections done on different days with
different cells with different complexes.
Assay for Luciferase Expression--
Harvested cells were
homogenized in lysis buffer (Promega), incubated for 15 min, and
centrifuged at 12,000 × g for 5 min, and the
supernatants were collected for assay. Aliquots (20 µl) were analyzed
for luciferase activity as described previously (23) and normalized for
protein content by the Bradford method (Bio-Rad). Results, expressed as
the integrated light units/mg protein, are the averages of duplicate samples.
Animals--
SEC-R ligands were based on the binding sequence of
human
1-antitrypsin but bind to receptors of mice and
rats as well as humans. SEC-R-directed DNA complexes containing the
optimal C105Y conjugates examined in vitro (11 ligands/DNA
molecule) were injected into the tail vein of anesthetized adult
C57/BL6 (Charles River Inc.) mice (8 weeks old, ~25 g). Complexes
contained 15 µg (50-60 µl) of the plasmid CMV lacZ II
bound to the optimal 9.7- or 53.7-kDa poly K-ligand conjugate (11 ligands/DNA plasmid molecule). Preliminary experiments showed no
advantage to increasing the dose to 25 µg DNA. Controls included
animals injected with a 1 M NaCl solution alone or with the
plasmid CMV lacZ II condensed with unconjugated poly K (9.7 or 53.7 kDa). Immediately following injection, there was 12% mortality
in all groups of animals injected with solutions containing 1 M NaCl due to apnea. Animals were sacrificed 2, 4, 16, and
30 days after treatment, and tissues were removed for analysis.
Preliminary tissue surveys (including the lung, liver, spleen, brain,
and kidney) showed that the highest activity was observed in lung and
spleen, so we examined these organs in this study. Examination of the
cellular distribution of lacZ activity in these tissues
showed that activity was evident only in macrophages. No
lacZ activity was detected with
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside staining
in the lungs or spleens of animals injected either with NaCl only or
with unconjugated poly K-DNA complexes. The animal research protocol
was reviewed and approved by the Case Western Reserve University
Institutional Animal Care Committee.
Assay for
-Galactosidase Activity--
Lungs and spleens of
animals were immediately frozen in liquid nitrogen and stored at
80 °C until homogenized in lysis solutions containing 12 mM leupeptin, 20 mM phenylmethylsulfonyl
fluoride, and 0.1% Triton X-100 in a phosphate-buffered saline
solution. Homogenates were incubated at 48 °C for 1 h to
inactivate endogenous
-galactosidase activity. Triplicate aliquots
were added to 200-µl reaction buffer (Tropix, Bedford, MA) and
incubated at room temperature for 1 h. 300 µl of light emission
accelerator (Tropix, Bedford, MA) was added, and the reaction was
monitored for 10 s in a luminometer.
DNase Protection Assay--
10-µg samples of pGL3 DNA were
condensed with either the 9.7- or 53.7-kDa optimal conjugates (11 ligands/DNA plasmid molecule) and incubated with DNase I (Boehringer
Mannheim) for 10 or 30 min in phosphate buffer containing
Mg2+. Samples were heated for 10 min at 80 °C to
inactivate the enzyme. The DNA was precipitated in a 75% ethanol/10%
NaCl solution, dried, and solubilized in water. Free DNA, in the
presence of ligand and sulfo-LC SPDP, was also digested for 10 and 30 min. Treated DNA in all treatment groups was subjected to agarose gel
electrophoresis to examine DNA integrity.
Statistical Analysis--
Data are expressed as the mean ± S.E. Statistical analyses of treatment groups were assessed using a
general linear modeling utilizing standard multiple regression
techniques (24). Residual analyses were conducted to assess validity of
model assumptions, including normality and homoscedasticity.
Logarithmic transformation (24) of the data was used to achieve
satisfactory conformity with model requirements. This modeling approach
was utilized for all analyses, including those related to the
evaluation of random substitution, where the data were considered as a
whole, and for specific experimental modalities to assess the
relationship between poly K size and the time of evaluation of
response. The same approach was applied to the subset of the data from
the dilution experiments that corresponded to concentrations of ligand
to DNA molecule common to the two ligands and also separately to an
extended series dilution for each ligand appropriate to the pattern of
responseof that ligand. Animal data are expressed as means ± S.D.
and evaluated by a nonparametric analysis of variance using the
Kruskall-Wallis analysis of variance of Ranks test (25).
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RESULTS |
NMR Analysis of the C105Y and C1315 Peptides--
Construction of
the protein conjugates of 9.7 and 53.7 kDa (average molecular mass)
poly Ks to the C105Y and C1315 peptides was monitored by NMR, both at
the step of sulfo-LC SPDP modification of poly K, and at the step of
conjugation of the peptides to modified poly K. This procedure enabled
us to calculate the ratio of poly K to linker to peptide. Key to the
analysis is the presence of unique aromatic proton resonances for both
the sulfo-LC SPDP conjugated to poly K and for the peptides conjugated
to SPDP-poly K. Proton NMR of sulfo-LC SPDP conjugated to poly K (10%
sulfo-LC SPDP mole ratio) produced three resonances that appear at 7.4, 7.9, and 8.5 ppm representing the four aromatic protons (H3, H4+H5, and H6, respectively) of the sulfo-LC SPDP 2-sulfhydryl pyridine group (26). Based on the molar ratios of poly Ks to sulfo-LC SPDP during the
coupling reaction calculated from five experiments on eight conjugates,
we estimate that this reaction was, on average, 77% efficient,
independent of poly K chain length.
1H NMR analysis of the C1315-poly K and C105Y-poly K
conjugates confirmed conjugation with the peptides has occurred by the disappearance of the sulfo-LC SPDP aromatic protons and by the appearance of new aromatic proton resonances in the region of 7-9 ppm
from aromatic residues in the ligand peptides.
Integration of conjugate proton resonances provided actual ratios of
poly K to linker to peptide. Both peptides had similar coupling
characteristics. For reactions where 0.1, 0.5, and 1.5% peptide was
added to poly K, 0.017% (1 in 5882 lysine residues modified), 0.097%
(1 in 1039 residues modified), and 0.27% (1 in 370 residues modified)
actually reacted, respectively. Modification with peptide was, on
average, 20% efficient. In eight different conjugates of each of the
poly Ks used, the chain length of poly K did not affect this efficiency
of coupling. Thus, we were able to obtain conjugates substituted to the
same extent with either linker or ligand on a per lysine residue basis
that differed only in poly K chain length.
Structure of Gene Transfer Complexes by Electron Microscopy and
AFM--
We examined the complexes by electron microscopy and AFM.
Solutions used to make complexes contained no structures, although DNA
was seen as rope-line strands. We measured electron microscopy photographs of 50 particles from each of eight complexes for each poly
K size and found DNA condensed with shorter average length poly K (9.7 kDa) formed complexes significantly larger than those constructed with
longer poly K (53.7 kDa) with the same substitution rate (24 ± 0.8-nm versus 17 ± 0.9-nm diameter, p < 0.01) (Fig. 1). The complexes appeared
spheroidal by rotary shadowing.

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Fig. 1.
Electron micrographs of SEC-R ligand
containing DNA complexes. Complexes formed using high salt
conditions (~1 M) were diluted 10-fold and immediately
pipetted onto a 1,000-mesh electron microscope carbon grid, fixed,
blotted, and stained with 0.04% uranyl acetate. Plasmid was SV40 pGL3
control (5.22 kb). The protein portion of the complex was: 53.7-kDa
poly K containing 3.5% sulfo-LC SPDP and 0.017% C105Y ligand
(A) and 9.7-kDa poly K containing 3.5% sulfo-LC SPDP and
0.017% C105Y ligand (B). Bar, 100 nm.
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The same conjugates were studied by AFM as well, which allowed us to
analyze both dry and hydrated complexes and also to evaluate their
three dimensional structure. In dried samples (50 particles), longer
length poly K (53.7 kDa, Fig.
2A) condensed DNA to particles averaging 25 ± 1.9 nm in diameter, whereas the shorter chain poly K condensed DNA into particles 39 ± 2.5 nm in diameter (9.7 kDa, p < 0.05; Fig. 2B). All complexes were
similar in height (16 ± 0.4 nm; Fig. 2). Images of hydrated
complexes were less distinct, and complexes appeared larger (~50-60
nm in diameter and ~20 nm in height), but complexes made with long
chain poly K appeared smaller than those with short chain poly K (data
not shown).

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Fig. 2.
Atomic force microscopy images of dried DNA
complexes on mica chips. Immediately after formation of DNA
complexes, a drop of the solution (1:100 dilution) was applied the
surface of a 2 × 2-cm Mica wafer. These samples were then dried
for 3 h and scanned by a Nanoscope III atomic force microscope.
DNA was SV40 pGL3 control plasmid (5.22 kb), and condensation proceeded
appropriately. The protein portion of the complex was: 53.7-kDa poly K
containing 3.5% sulfo-LC SPDP and 0.017% C105Y ligand (A)
and 9.7-kDa poly K containing 3.5% sulfo-LC SPDP and 0.017% C105Y
ligand (B). Gray scale indicates height of
complexes. Bar, 100 nm.
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Effect of Chain Length on DNA Protection--
As examined by gel
electrophoresis, the pGL3 plasmid was more protected from DNase
degradation when condensed with longer poly K (up to 10 min at
37 °C) than DNA condensed with shorter poly K or free DNA (data not
shown). Both long and short poly Ks failed to protect DNA against
degradation after 30 min of treatment with DNase I.
Experimental Strategy--
To test the contribution of the chain
length of the poly K molecule to the intensity and duration of gene
expression, we used two different approaches, random substitution and
ligand dilution, which generally gave concordant results. To focus this
comparison on poly K length, we first report on the selection of the
better ligand and the best ratio of ligands to DNA.
Comparison of Receptor-directed Complexes and Controls--
All
transfections with complexes containing any of the poly K linked to
ligand produced significantly greater luciferase expression (p < 0.01) than transfections with DNA alone
(n = 18), DNA condensed with unmodified poly K (all
sizes) in the presence of linker (n = 36), and DNA
condensed with linker modified poly K (n = 24). Transfections with Lipofectin-DNA complexes, used as a positive control, resulted in peak activity 8-10-fold lower than the average peak luciferase activity obtained with ligand-containing complexes (~107 integrated light units/mg protein; Fig.
3). Expression from Lipofectin transfection was indistinguishable from background by 10 days.

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Fig. 3.
Effect of poly K size on reporter gene
expression. HuH7 cells were transfected with 1.11 pmol of
complexed DNA, and luciferase activity was assayed at 2, 4, 10, and 16 days later. Nine experiments were conducted with each of the
constructed conjugates. In all 36 conjugates were examined, 12 with
each of the different size poly Ks (6 with each peptide).
Representative figures for 18 of those conjugates are demonstrated
above. Graphed values represent averages of the experiments conducted
for each of the conjugates (n = 9), and error
bars show S.E. A, luciferase activity obtained from
cells transfected with two different size poly Ks substituted with
7.8% linker and 0.27% C105Y ligand. B, luciferase activity
obtained from cells transfected with two different size poly K
substituted with 3.5% linker and 0.017% C105Y ligand. C,
luciferase activity obtained from cells transfected with various
control mixtures lacking receptor ligand. In all nine experiments with
each of the conjugates displayed above, luciferase activity was
significantly greater (p < 0.01) than cells
transfected with DNA alone or unmodified poly K condensed DNA.
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Random Substitution Experiment--
Complexes constructed with the
53.7-kDa poly K produced peak expression at 4-10 days post
transfection, whereas complexes containing the 9.7-kDa poly K peaked at
2 days but declined rapidly. Two days post transfection, complexes with
the shorter poly K produced, on average, 10-fold higher expression than
the longer poly K. At 4-10 days, the longer poly K complexes produced
greater expression. At 16 days, the longer poly K resulted in
significantly higher expression than the shorter poly K (C105Y,
p = 0.0001 and C1315, p = 0.0162). Fig.
3 shows a representative example for the most and least substituted
complexes. We compared the effects of C1315 and C105Y on intensity of
duration of reporter gene expression. Both peptides gave comparable
intensity of expression, but the C105Y peptide containing complexes
produced longer lasting expression when compared with the C1315 peptide
over a broad range of substitution patterns (p < 0.01).
To further examine this phenomenon, we performed five experiments with
each of the 53.7-kDa poly K constructs and extended the time course to
40 days (Fig. 4). Although in these
experiments there is no significant difference between the two peptides
at day 2 or 18, all 53.7-kDa constructs containing the C105Y peptide expressed higher levels of luciferase at days 25 and 40 post
transfection than constructs with C1315 (n = 60, p < 0.01). No excess toxicity (as assessed by whole
cell protein measurements) was observed for either peptide.

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Fig. 4.
Effect of ligand type on the duration of
reporter gene expression. HuH7 cells were transfected with 1.11 pmol of complexed DNA, and luciferase activity was assayed at 2, 18, 25, and 40 days post transfection. Six experiments were conducted with
each of the 53.7-kDa poly K constructed conjugates. In all, 12 conjugates were examined, 6 with each of the different peptides. The
values plotted represent averages of the experiments conducted for each
of the conjugates (n = 6). A, luciferase
activity obtained from cells transfected with all the 53.7-kDa poly K
conjugates with the C105Y ligand. B, luciferase activity
obtained from cells transfected with all the 53.7-kDa poly K conjugates
with the C1315 ligand.
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Ligand Dilution Experiment--
Due to the complexity of the
random substitution results, we designed a simpler experiment to
address the optimal ligand to DNA ratio. We assumed that all of the
conjugate added to DNA during the condensation step complexes with the
plasmid. The size of the plasmid (5.22 kb) gives the number of negative
charges. Because we know the ligand to lysine residue ratio, we can
estimate the number of ligands/DNA molecule once the lysines have
completely neutralized the 10440 negatively charged phosphate groups on
each molecule of DNA. For the 53.7-kDa poly K constructs substituted once with ligand, a maximum of ~43 ligands would interact with each
DNA molecule; for the 9.7-kDa poly K substituted once, a maximum of
~290 ligands would interact. This ligand number could be reduced by
dilution with unmodified poly K.
Initial experiments with each of the poly K sizes included complexes
containing 5-40 ligands (C105Y and C1315)/DNA molecule. For the C105Y
ligand, optimal complex preparations contained 8-14 ligands/DNA
molecule (Fig. 5). Six separate
experiments conducted with the 53.7-kDa poly K constructs demonstrated
that all complexes except those containing 40 ligands/plasmid produced
expression, which peaked at 4 days post transfection and remained
higher (p < 0.01) than background for the duration of
the experiments (16 days). For the C1315 ligand, 20-35 ligands/DNA
were required for optimal expression, but complexes with 20-25
ligands/DNA molecule retained expression best.

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Fig. 5.
Ligand dilution required for efficient
delivery and expression of the pGL3 plasmid complexed with the 53.7-kDa
poly K. 53.7-kDa poly K-ligand conjugates containing one
ligand/poly K were diluted with the unmodified 53.7-kDa poly K to
varying extents and used to condense DNA (n = 6). The
number of ligands represents the theoretical number of ligands/DNA
molecule at charge neutralization. A, peak expression (4 days) observed for complexes containing the C105Y ligand. B,
prolonged expression (16 days) for complexes containing the C105Y
ligand. C, peak expression (4 days) observed for complexes
containing the C1315 ligand. D, prolonged expression (16 days) observed for complexes containing the C1315 ligand.
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Complexes constructed with the 9.7-kDa poly K (Fig.
6) produced expression that peaked at 2 days and then rapidly declined. The optimal ligand ratio to DNA
molecule was similar to long chain poly K, for C105Y (~8-14 ligands)
or C1315 ligand (~20-35 ligands). Thus, for these experiments, as
for the random substitution experiments, short length poly K complexes
had high initial expression with rapid decline, and longer chain poly K
complexes had expression that peaked later and persisted longer.

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Fig. 6.
Ligand dilution required for efficient
delivery of plasmid DNA complexed with the 9.7-kDa poly K. Conjugates of the 9.7-kDa poly K to the SEC-R ligands C105Y and C1315
(one ligand/poly K) were diluted with unmodified 9.7-kDa poly K to
varying extents and used to condense the pGL3 plasmid
(n = 6). Number of ligands represents the theoretical
number ligands/DNA molecule at charge neutralization. A,
peak luciferase expression (2 days) observed for complexes containing
the C105Y ligand. B, prolonged expression (16 days) for
complexes containing the C105Y ligand. C, peak expression (2 days) observed for complexes containing the C1315 ligand. D,
prolonged expression (16 days) observed for complexes containing the
C1315 ligand.
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Like the random substitution experiments, the ligand dilution
experiments showed that C105Y gave better duration and no less intensity than C1315. Sixteen days post transfection, conjugates containing 5-20 C105Y ligands/DNA molecule and the longer or shorter poly K produced higher expression (p = 0.0001) than
those containing the C1315 ligand. When comparing the effect of poly K
length on duration of expression (at 16 days), we found that complexes
containing the 53.7-kDa poly K produced longer duration of expression
than those containing the 9.7-kDa poly K (p = 0.0005).
Furthermore, ligand dilutions between 5 and 20 ligands/DNA molecule
produced longer duration of expression than complexes containing 40 ligands/DNA molecule (p = 0.0002). Conversely, at 2 days, complexes containing the shorter poly K produced higher
expression than those containing the longer poly K
(p = 0.0001).
Effect of Poly K Length on in Vivo Gene Transfer--
To test
whether our in vitro model for SEC-R-mediated gene transfer
predicts, in principle, the in vivo response, we injected via the tail vein C57/BL6 mice with DNA complexes containing the CMV
lacZ II plasmid (10.8 kb) bound to C105Y conjugated to
either the 53.7- or 9.7-kDa poly K (with 11 ligands/DNA plasmid
molecule). C105Y and the number of ligands/DNA molecule were chosen to
maximize long term expression based on in vitro data.
-Galactosidase activity in the lung and spleen of these animals
(n = 4-5 animals for each treatment group at each time
point) injected with DNA complexes containing the C105Y/9.7-kDa poly K
peaked 4 days after injection (different from salt, p < 0.01, and unsubstituted poly K complex injected control animals;
53,751 integrated light units/mg protein ± 9,006 in lung and
42,474 integrated light units/mg protein ± 6,323 in spleen;
p < 0.001), was declining by 16 days post treatment, and no longer differed from background. Animals injected with the
C105Y-53.7-kDa poly K conjugate-DNA complexes had peak expression at 16 days, which had disappeared by 30 days (Fig.
7). Gene expression in animal groups
treated with the C105Y-53.7-kDa complexes differed statistically from
control animals treated with 1 M salt (p < 0.001) or complexes formed with unsubstituted poly K and DNA at both 4 and 16 days (p < 0.01). At 16 days, expression from
the C105Y-53.7-kDa poly K complexes also significantly exceeded that from complexes containing the C105Y-9.7-kDa poly K. Fig. 7 demonstrates in vivo gene expression using both poly K lengths and the
controls. Animals treated with complexes containing DNA and
unconjugated poly K did not differ from salt injected animals at
any time point.

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Fig. 7.
In vivo
-galactosidase expression in mice treated with DNA
complexes containing the 53.7- or 9.7-kDa poly Ks. Optimal
conjugates selected from in vitro studies were used to
deliver the CMV lacZ II plasmid to mice in vivo.
Asterisks represent data points that are statistically
different (based on p value) from animals injected with 1 M NaCl only (squares) or complexes of plasmid
with unconjugated poly K of the respective length
(diamonds). Double asterisks represent points
that differ between C105Y-9.7-kDa poly K-DNA (white circles)
complexes and C105Y-53.7 kDa poly K-DNA (black circles)
complexes. A, the effect of poly K length on expression
measured in the lungs of animals (n = 4-5 for each
time point). B, the effect of poly K length on expression
measured in the spleen of animals (n = 4-5 for each
time point).
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DISCUSSION |
The composition of the protein portion of receptor-targeted gene
transfer protein-DNA complexes profoundly affects the intensity and
duration of gene expression, both in vitro and in
vivo. In vitro the number of ligands/DNA molecule, the identity of
the ligand, and the chain length of poly K all have strong effects on
intensity and duration of gene expression, but the most striking effect
was that of poly K chain length on the duration of expression. Using
the ligand and number of ligands/DNA molecule found to be optimal in
the cell culture system, we tested the effect of poly K chain length on
in vivo expression. Complexes made with long chain poly K produced much
more protracted gene expression in vivo than complexes
prepared with short chain poly K.
Optimization of the SEC-R-directed gene transfer for in HuH7 cells
resulted in greatly increased reporter gene expression (relative to
Lipofectin control) compared with our prior report (9). Activity in the
best complexes tested in the present study was about 20-fold higher
than activity produced by the same amount of plasmid DNA delivered with
the Lipofectin reagent; in our previous report maximal activity was
less than the Lipofectin control. Increased peak activity indicates
increased efficiency, which has been a problem for most nonviral
strategies. At the same time, duration of activity could be greatly
prolonged in vitro by informed selection of the specific
receptor ligand (C105Y versus C1315), the number of
ligands/plasmid DNA molecule (over 2-40 ligands), and the chain length
of poly K used to condense the DNA. It is likely that the number of
ligands and the identity of the ligand entrains routing within the
cell, with the best complexes promoting routing into the endosomal
compartment rather than to the lysosomal degradative pathway. For
another receptor, the epidermal growth factor receptor, high number of
ligands promotes lysosomal trafficking, whereas fewer ligands promote
endosomal recycling (27), so fewer ligands might well entrain a more
favorable trafficking pattern, delaying the destruction of delivered
DNA. In addition, different ligands for some receptors, such as the
transferrin receptor, may be trafficked differently (28). A similar
mechanism may account for the differences in duration of expression for
complexes containing the two SEC-R ligands, which have very similar
affinity for the receptor and very similar initial transgene expression.
In vitro, the expression of the firefly luciferase reporter
gene persisted (within a log maximum) in HuH7 cells for at least 40 days for some complexes that contained the long chain poly K-ligand
conjugates. Because the half-life of the luciferase protein in
mammalian cells is 3 h (29) and we observed rapid decline in
luciferase activity in HuH7 cells transfected using Lipofectin (thus,
HuH7 cells do not retain high levels of luciferase activity, however
the gene is delivered), protracted luciferase expression from complexes
made with long chain poly K probably represents continuing
transcription of plasmid DNA. In an in vitro assay, we did
not observe transcription from plasmid DNA compacted with either short
or long chain poly K, so it is likely that in vivo, plasmid
DNA separates from poly K before transcription. These considerations
suggest that the prolonged expression results from retention of plasmid
DNA complexed with poly K for a longer period of time for complexes
containing long chain poly K than for the short chain. Condensation
with poly K protects DNA against degradation (4), and better protection
is afforded by long chain poly K than short chain in in
vitro assays. Thus, we speculate that plasmid DNA is retained
within the cell for both complexes (because both have better
persistence of expression than Lipofectin) with gradual degradation,
which is resisted longer by complexes containing long chain poly K. Whether the complexes reside in membrane-bound cytoplasmic
compartments, the cytoplasm, or the nucleus is not yet clear. It is
also possible that the less tightly compacted complexes with the
shorter poly K allow better access for the RNA polymerase to the DNA
and accounts for the higher initial expression. In such a scenario,
transgene expression would depend on a dynamic relationship between the
availability of the DNA for transcription and the rate of its
subsequent degradation.
Transgene activity persists better in vitro (40 days) than
in vivo (less than 30 days), even for genes delivered using
complexes containing long chain poly K. There are probably several
reasons for this difference. In vitro, only processes within
the transfected cell account for decay of transgene activity, whereas
in vivo, immune processes can be recruited to destroy cells
expressing proteins not recognized as "self." Bacterial
-galactosidase has been shown, in and of itself, to incite cytotoxic
lymphocyte responses in vivo, and therefore, cells
expressing this protein are preferentially targeted and destroyed (27).
This is probably one mechanism of extinguishing
-galactosidase
activity. In addition, the HuH7 cells transfected in vitro
are immortal but fail to grow and divide after about 12-14 days under
our culture conditions if they are not subcultured. In vivo,
normal cell turnover of transfected cells may limit duration of expression.
Complexes containing a minimal number of ligands that specifically
deliver exogenous DNA to receptor bearing cells might be less
immunogenic than more heavily substituted complexes. DNA (30) and
poly-L-amino acids (31) have been found to be relatively nonimmunogenic. Thus, possible immunogenicity of receptor-targeted complexes in vivo may depend on the ligand portion and, in
part, its abundance. The abundance of SEC-R in lung, liver, and brain (14), all of which might be potential target tissues for therapeutic gene transfer in common inherited (e.g.
1-antitrypsin deficiency) or acquired (e.g.
Alzheimer's disease) disorders, has made it a desirable target for
receptor-mediated gene therapy. The development of optimal complexes
that produce high level gene expression for short (9.7-kDa poly K
conjugates) or longer (53.7-kDa poly K conjugates) periods of time will
be useful in achieving optimal therapeutic effects.