Galactose-terminated (asialo-) glycoproteins (ASG) (
)are internalized via the asialoglycoprotein receptor
(ASGR), which is expressed in large numbers only in hepatocytes.
Following endocytosis, the endosomal vesicles containing the ligand are
translocated to lysosomes where the ligands are degraded. During this
translocation, the ligand is uncoupled from the receptor, which returns
to the cell surface and is reutilized(1) . Because of the high
level of expression of ASGR in hepatocyte surface membranes,
ASGR-mediated endocytosis has been utilized for targeted delivery of
macromolecules, including DNA, to the liver(2) . For this
purpose, ASGs are conjugated to polycations, such as polylysine. Under
appropriate conditions, these conjugates form a soluble complex with
DNA. After intravenous infusion, the ASG-polycation-DNA complex is
internalized by hepatocytes via ASGR-mediated endocytosis. As expected,
DNA internalized by this pathway is rapidly degraded, presumably in
lysosomes. However, a small fraction of the DNA apparently reaches the
nucleus and is expressed transiently(3, 4) . In an
effort to prolong the transgene expression, 66% hepatectomy has been
performed after DNA delivery. This procedure results in persistence and
expression of the endocytosed DNA for many
weeks(5, 6) . The great majority of the persisting DNA
exists in a pool of cytoplasmic vesicles (7) as undegraded
input plasmid(6, 7) . The mechanism of persistence of
the endocytosed DNA in hepatocellular cytoplasm is not clear. The wave
of hepatocellular mitosis that occurs 16-48 h after partial
hepatectomy cannot explain this persistence, because a great majority
of the endocytosed DNA is rapidly degraded in nonhepatectomized
recipients within the first 4 h after DNA
delivery(6, 7) .
We have observed recently that the
microtubular network in a significant proportion of hepatocytes is
transiently disrupted after 66% hepatectomy(8) . Because intact
microtubules are required for translocation of endosomes to
lysosomes(9) , it is possible that the early disruption of
microtubules after partial hepatectomy may play a role in transgene
persistence after this procedure. Based on these observations, we
hypothesized that transient pharmacological disruption of microtubules
may also prolong the persistence and expression of DNA endocytosed into
hepatocytes. In this paper, we have used
bilirubin-UDP-glucuronosyltransferase-deficient homozygous Gunn rats (10) to evaluate this hypothesis. Two plasmids, one expressing
bacterial chloramphenicol acetyl transferase and another expressing
human bilirubin-UDP-glucuronosyltransferase-1, were transferred by
ASGR-mediated endocytosis in vivo. The effect of microtubular
disruption by colchicine administration on the persistence and
expression of these gene were determined.
MATERIALS AND METHODS
Animals
Homozygous Gunn rats and congeneic
normal Wistar-RHA rats of both genders (150-200 g) were obtained
from our colony at the Albert Einstein College of Medicine. The rats
were maintained on standard laboratory rat chow in a 12-h light/dark
cycle. All animals received humane care in accordance with guidelines
of the National Institutes of Health and the Animal Research Committee
of the Albert Einstein College of Medicine.
Chemicals
An anti-rat brain
-tubulin
monoclonal antibody, an fluorescein isothiocyanate-conjugated
anti-mouse IgG antibody, and colchicine were purchased from Sigma.
Polyvinyl pyrrolidone sheets (Immobilon) for immunotransblot were
obtained from Millipore (Bedford, MA). A plasmid, pSV2-CAT, containing
the coding region of bacterial chloramphenicol acetyltransferase (CAT)
driven by the SV40 large T antigen promoter was a gift from Dr. David
A. Shafritz. A plasmid containing the coding region of human
bilirubin-UDP-glucuronosyltransferase-1 (pSVK3-hBUGT
), also
driven by the SV40 large T antigen promoter, was constructed as
reported(11) .
Synthesis of the ASG-Polylysine Conjugate and Formation
of the DNA-Carrier Adduct
Asialoorosomucoid was produced by acid
hydrolysis (12) of orosomucoid isolated from pooled human serum (13) and covalently linked with polylysine (Sigma; average
molecular weight, 59,000) as described(2, 7) . The
plasmid DNA was added to the ASG-polylysine conjugate at a 1:2 ratio
(w/w) to produce a soluble adduct. At this proportion, the
electrophoretic mobility of the plasmid on agarose gel electrophoresis
was fully inhibited(2) . The complex was filtered through a
0.2-µm filter and was stored at 0-4 °C for up to 1 week
before infusion into rats.
Determination of the Dose of Colchicine and Time Course
of Its Action
In initial experiments we determined the maximum
tolerated dose of colchicine in Gunn rats and congeneic normal Wistar
RHA rats. In both groups, significant mortality was observed with a
dose of 2 mg/kg of body weight injected intraperitoneally. Therefore,
we limited the maximum dose to 1 mg/kg of body weight for subsequent
studies. To determine the minimum dose required for microtubular
disruption, colchicine (0.25-1.0 mg/kg of body weight) was
dissolved in normal saline and injected intraperitoneally (4 rats in
each group). Controls (n = 4) received normal saline
intraperitoneally. The rats were anesthetized with ether, and the
livers were prepared for immunofluorescence confocal microscopy as
described below. To determine the time course of action of colchicine,
we injected 0.75 mg of colchicine (which was the minimum dose required
for extensive microtubular disruption) into five groups of rats (two in
each group), which were sacrificed for examination of hepatocellular
microtubules 1, 2, 4, 6, 24, and 48 h after colchicine injection.
Infusion of Colchicine and DNA-Carrier Adducts
The
DNA-carrier complex (100 pmol DNA in 5.0 ml), was infused into tail
veins of Gunn rats anesthetized with ether. Because the disruption of
microtubules results in the depletion of cell surface ASGR over time,
in preliminary studies we determined the optimum temporal relationship
between administration of colchicine and infusion of the DNA-carrier
complex. Injection of colchicine 30 min before DNA administration
resulted in a combination of efficient internalization of the DNA by
the liver and its persistence in hepatocytes (data not shown). For
determination of the effect of gene therapy on serum bilirubin levels,
six Gunn rats were injected with the carrier-pSVK3-hBUGT
without colchicine pretreatment, and six others were injected
with the DNA-carrier complex 30 min after colchicine pretreatment. Fourteen other Gunn rats in each group were used for determining the
time course of DNA persistence, human
bilirubin-UDP-glucuronosyltransferase-1 expression,
bilirubin-UDP-glucuronosyltransferase activity, and bile pigment
analysis. For these studies, two rats in each group were killed at
various intervals. For determination of hepatic CAT expression, 42
Wistar-RHA rats were used in the colchicine-treated group, and 14
Wistar RHA rats were used in the group that received the DNA without
colchicine pretreatment. Three rats were killed at various time points
for harvesting the livers for CAT assay.
Preparation of the Liver and Confocal Immunofluorescence
Microscopy
Fixation of the liver was carried out in control
animals (no colchicine administration) and at various doses or time
intervals after colchicine injection as described above. Technical
details of tissue preparation, immunofluorescence staining, and
confocal microscopy have been described previously(8) . In
brief, the rats were anesthetized with ether, and the livers were fixed
by in situ perfusion with 4% paraformaldehyde. Processing for
a given series were completed within a 24-h period. Because
microtubules disassemble at low temperatures, care was taken to fix the
livers in situ at 37 °C. After fixation, all procedures
were carried out at room temperature. Tissue sections (45 µm) were
prepared with an Oxford Vibratome (Ted Pella, Testin, CA). The tissue
sections were permeabilized by exposure to 0.1% Triton X-100 for 1 min
and then reacted with an anti-
-tubulin antibody and subsequently
with an anti-mouse IgG-fluorescein isothiocyanate conjugate. Confocal
microscopy was carried out with a Bio-Rad MRC 600 scanning confocal
microscope outfitted with a krypton/argon laser using a Nikon planapo
60
N.A. 1.40 objective. Coded sections were scanned at
0.5-µm intervals to a maximum depth of 3-6 µm, with the
observers not aware of the experimental conditions. When comparing
control and experimental sections, observations were made at the same
depth. The laser intensity and all image collection parameters were
held constant throughout and were checked for stability at the end of
the survey of the sections. Between 20 and 30 fields of each section
were scanned, and an overall assessment of the extensiveness of the
fluorescently labeled microtubular network was made. At the end of the
examination of a series of sections, the code was broken.
DNA Extraction and Southern Blot Analysis
Liver
samples or isolated cells were homogenized in 10 mM Tris-HCl,
pH 8.0, containing 100 mM NaCl and 1 mM disodium EDTA
in a glass/teflon homogenizer. Proteinase K and sodium dodecyl sulfate
were added at 1 mg/ml and 10 mg/ml, respectively, and the mixture was
incubated at 37 °C for 18 h. After extraction with
phenol/chloroform (1:1, v/v) and chloroform, DNA was precipitated from
the aqueous phase with 2 volumes of isopropanol. Isopropanol was
evaporated in reduced pressure, and DNA was resuspended in 10 mM Tris-HCl, pH 7.4, containing 1 mM disodium EDTA. The
plasmid was linearized by digestion with the restriction enzyme ApaI, which has a single recognition site on the plasmid
pSVK3-hBUGT
(11) . After electrophoresis on 0.8%
agarose gels, the DNA was transferred to Genescreen membranes (DuPont
NEN), and Southern analysis (14) was performed using a
P-labeled probe directed toward the unique 5` region of
human bilirubin-UDP-glucuronosyltransferase-1(15) . Band
intensities were determined by densitometry of autoradiograms.
Assay of CAT Activity
CAT activity in liver
homogenates was assayed in triplicate using
[
C]chloramphenicol as a substrate and thin-layer
chromatographic analysis as described (16) .
Immunotransblot Studies
After bile collection,
livers were removed, and 20% homogenates were prepared in 0.25 M sucrose in 10 mM Tris-HCl, pH 7.8, containing 0.1 mM EDTA. The homogenates were centrifuged at 800
g for 10 min. The supernatant was collected and centrifuged at
12,000
g for 15 min. This supernatant was centrifuged
at 105,000
g for 60 min. A 10% suspension of the
pellet (microsomes) was made in 9 ml of the homogenization buffer/g of
wet weight of the pellet. The microsomal suspension was used for
immunoblot studies and bilirubin-UDP-glucuronosyltransferase assay (see
below).Samples of the microsomal suspension, containing 100 µg
of protein were subjected to SDS-10% polyacrylamide gel
electrophoresis, and immunotransblot studies were performed using an
antibody specific for human bilirubin-UDP-glucuronosyltransferase-1 and
an anti-mouse IgG antibody conjugated with alkaline
phosphatase(11) . Band intensities were determined by
densitometry.
Bilirubin-UDP-glucuronosyltransferase
Activity
UDP-glucuronosyltransferase activity toward bilirubin
in the microsomal suspensions was assayed at 5 mM uridine
diphosphoglucopyranuric acid by high pressure liquid chromatographic
(HPLC) analysis of the products, as we have described
previously(18) . Formation of bilirubin glucuronides was
proportional to protein concentrations in the assay mixture and
duration of incubation.
Analysis of Bilirubin Glucuronides in Bile by
HPLC
Pigments excreted in the bile were analyzed by
reverse-phase HPLC using a Waters C-18 column as
described(19) . Bilirubin mono- and diglucuronide were
quantified by electronic integration of peak areas.
Serum Bilirubin Concentrations
Serum total
bilirubin concentrations were determined using diazotized
ethylanthranilate in the presence of dimethyl sulfoxide as an
accelerator as previously established(20) , and the identity of
the pigment as unconjugated azodipyrrole was established by thin-layer
chromatography(21) .
RESULTS
Dose of Colchicine and Time Course of Its
Effect
In control sections, a delicate and extensive system of
microtubules was seen throughout the hepatocytes and nonparenchymal
cells. Because the degradation of the majority of the internalized DNA
occurs within the first hours after
endocytosis(6, 7) , we wanted to determine the dose of
colchicine that is adequate for microtubular disruption shortly after
its administration. Therefore, the dose-response relationship was
determined 2 h after colchicine injection. Disruption of microtubules
was minimal at a dose of 0.25 mg/kg of body weight (Fig. 1B). The microtubules were significantly
disrupted at higher doses (Fig. 1, C, D, and E). Microtubular disruption was nearly maximal with 0.75 mg/kg
of colchicine (Fig. 1D); this dose was used for
subsequent experiments. At 0.75 mg/kg, a significant decrease in
microtubular structure in a majority of hepatocytes was noted within 1
h, and maximum microtubular disruption was observed in 2 h. By 24 h,
the microtubular network had partially regenerated, and 48 h after
colchicine administration, the microtubular network was fully
regenerated.
Figure 1:
Effect of colchicine
on hepatocyte microtubular network. 2 h after the administration of
colchicine or normal saline (control), rats were anesthetized and liver
were fixed in situ by perfusion with paraformaldehyde as
described in the text. Immunofluorescence studies were performed by
scanning confocal microscopy using an anti-
-tubulin antibody. Two
sets of experiments are shown. Set 1 (upper row): A,
normal saline; B, 0.25 mg of colchicine/kg of body weight; C, 1 mg of colchicine/kg of body weight. Set 2 (lower
row): D, normal saline; E, 0.75 mg of
colchicine/kg of body weight.
Time Course of Degradation of pSVK3-hBUGT
Internalized via ASGR-mediated Endocytosis
To correlate
our studies of microtubule integrity with the degradation of
internalized DNA, we digested the DNA extracted from liver homogenates
with ApaI and performed Southern blot analysis using an
oligonucleotide probe specific for the unique 5` domain of human
bilirubin-UDP-glucuronosyltransferase-1 (Fig. 2). Densitometric
analysis of the Southern blots showed that in 20 min, 70-80% of
the infused DNA was internalized by the liver 20 min after injection,
representing about 30,000-35,000 copies of the plasmid per
hepatocyte. This calculation was based on our previous finding that 80%
of the DNA internalized by the liver is present in hepatocytes (7) . In control rats (without colchicine pretreatment), only
about 10% of the initial DNA load remained in the liver 4 h after
administration (Fig. 2A); by 24 h, the plasmid DNA was
undetectable by Southern blot. In contrast, in rats that were
pretreated with 0.75 mg of colchicine/kg of body weight, 30% of the
endocytosed plasmid was retained by the liver at 4 h (Fig. 2B). By 24 h after DNA administration, the
plasmid concentration had decreased to 8-12% of the level at 20
min. The DNA concentration remained nearly at this level for 8-10
weeks, after which it declined further and became undetectable in 14
weeks.
Figure 2:
Southern blot analysis of DNA internalized
by the liver. Liver samples were collected at various time points from
Gunn rats that were administered pSVK3-hBUGT
without (A) or after (B) pretreatment with colchicine. Liver
homogenates were treated with proteinase K and sodium dodecyl sulfate,
and DNA was extracted as described in the text. The DNA was digested
with the restriction enzyme ApaI, which has a single
recognition site on the plasmid pSVK3-hBUGT
. After
electrophoresis on 0.8% agarose gels, Southern analysis was performed
using a
P-labeled probe specific for human bilirubin
UDP-glucuronosyltransferase-1. Liver samples were collected at 4 h (lanes 1), 24 h (lanes 2); 2 weeks (lanes
3), 5 weeks (lane 4), 8 weeks (lane 5), 10 weeks (lane 6), and 14 weeks (lane 7). Each data point is
from a single rat, representative of three
experiments.
Hepatic CAT Activity
CAT activities in homogenates
of livers removed from rats at various intervals after infusion of the
plasmid-carrier complex are shown in Fig. 3. CAT activity was
undetectable in the liver samples at 20 min or 4 h after DNA
administration. At 24 h, CAT activity was detected at approximately 2.0
microunit/mg of protein in the rats that were not pretreated with
colchicine and 2.5 microunits/mg of protein in rats that received
colchicine injection 30 min before DNA administration; the difference
was not statistically significant (p > 0.2). Seven days
after DNA administration, CAT activity was undetectable in
nonpretreated rats but was detected at approximately 1.0 microunits/mg
of protein in the group that received colchicine. In the
colchicine-pretreated group, CAT activity persisted at approximately
this level for 8 weeks, after which it progressively declined and
became undetectable by the end of the study (14 weeks).
Figure 3:
Hepatic CAT activity. The DNA-carrier
complex was injected without colchicine treatment (
) or after
colchicine treatment (
). Liver tissues were collected at the
indicated time points, and CAT activity was determined as described in
the text. Each data point represents the mean ± S.D. of data
from three rats.
Expression of Human
B-UDP-glucuronosyltransferase
Expression of the
human bilirubin-UDP-glucuronosyltransferase-1 protein was determined by
immunotransblot using an isoform-specific anti-peptide antibody (Fig. 4). This antibody did not recognize any protein from
livers from untreated Gunn rats. In the group of rats that were
pretreated with colchicine, the immunoreactive human
bilirubin-UDP-glucuronosyltransferase-1 was detectable from 24 h to 10
weeks. In the nonpretreated group, the enzyme protein was detectable
only at the 24-h point after DNA administration.
Figure 4:
Immunoblot with an anti-human
bilirubin-UDP-glucuronosyltransferase-1. Livers were collected from
Gunn rats that received pSVK3-hBUGT
, and microsomal
fractions were prepared as described in the text. Microsomal
suspensions containing 100 µg of protein were subjected to SDS-10%
polyacrylamide gel electrophoresis, and immunotransblot studies were
performed using an antipeptide antibody specific for the unique region
of human bilirubin-UDP-glucuronosyltransferase-1 and an anti-mouse IgG
antibody conjugated with alkaline phosphatase. Results are shown from a
single representative experiment. For comparison, human liver
microsomes (50 µg of protein) were applied in lanes 1 and 5. A, liver microsomes from Gunn rats that received
pSVK3-hBUGT
without pretreatment with colchicine. B, liver microsomes from Gunn rats that received the DNA
following pretreatment with colchicine. Liver samples were harvested at
following intervals after DNA administration: Lanes 2 and 6, 4 h; lanes 3 and 7, 24 h; lanes 4 and 8, 2 weeks; lane 9, 8 weeks. Each data point
is from a single rat, representative of data from two
rats.
Bilirubin-UDP-glucuronosyltransferase
Activity
UDP-glucuronosyltransferase activity toward bilirubin
was detectable in liver microsomal fractions from both
colchicine-pretreated and nonpretreated groups at 24 h after
administration of pSVK3-hBUGT
(Table 1). At
subsequent time points, the enzyme activity was detectable in only in
the colchicine-pretreated group for up to 8 weeks after DNA
administration. For comparison, we determined
bilirubin-UDP-glucuronosyltransferase activity in human liver
microsomes. The specific enzyme activity in the hepatic microsomes of
colchicine pretreated Gunn rats was approximately 2-4% of that in
human liver microsomes (10-20 nmol/mg of protein/min).
Bilirubin Glucuronides Excreted in Bile
To
directly determine bilirubin-UDP-glucuronosyltransferase activity in vivo, we analyzed pigments excreted in the bile. The bile
of untreated Gunn rats (not shown) or Gunn rats treated with pSV2CAT (Fig. 5A) did not contain significant amounts of
bilirubin glucuronides, the majority of the pigments excreted in bile
being unconjugated bilirubin. 24 h after administration of
pSVK3-hBUGT
, significant amounts of bilirubin
monoglucuronide and small amounts of bilirubin diglucuronide were
detectable in the bile of rats from both the colchicine-pretreated and
noncolchicine-treated group. After this time, bilirubin glucuronides
were present only in the bile of rats from the colchicine-pretreated
group. 1-8 weeks (Fig. 5B) after the
administration of pSVK3-hBUGT
, the bile from this group
contained 20-40% of bilirubin glucuronides (predominantly
bilirubin monoglucuronide and a variable proportion of bilirubin
diglucuronide) excreted in normal rat bile. However, unconjugated
bilirubin accounted for nearly 60-80% of the total pigments
excreted in bile, whereas only 1-3% the pigments in normal rat
bile consisted of unconjugated bilirubin (not shown). From the 10th
week onward, only minor amounts of conjugated bilirubin were detectable
in the bile of the colchicine-pretreated Gunn rats (not shown).
Figure 5:
HPLC
of bile pigments. Gunn rats were provided with bile duct cannulae under
ether anesthesia and placed in restraining cages. Bile was collected at
the indicated times after DNA administration. Bile pigments were
separated by HPLC, identified from retention time by comparison with
authentic purified pigments, and quantified by integration of areas
under the peaks. Peak identifications are indicated. sf,
solvent front; BDG, bilirubin diglucuronide; BMG,
bilirubin monoglucuronide; UCB, unconjugated bilirubin. A, 4 h after pSVK3-hBUGT
administration following
colchicine pretreatment. B, 6 weeks after DNA administration
following colchicine pretreatment. C, 2 weeks after DNA
administration without colchicine pretreatment. Each bile sample is
from a single rat, representative of two in the
group.
Serum Bilirubin Concentrations
In the
colchicine-pretreated group, serum bilirubin concentrations declined by
25-35% in 2-4 weeks, remained at the reduced levels for
4-6 weeks, and then gradually returned to pretreatment levels by
14 weeks (Fig. 6). Without colchicine pretreatment, serum
bilirubin remained unchanged from the pretreatment levels.
Figure 6:
Serum
bilirubin concentrations. Blood samples were collected at the indicated
times, and serum bilirubin was determined as described in the text.
Each data point is the mean of six experiments ± S.E.
, DNA
without colchicine;
, DNA with
colchicine.
DISCUSSION
Translocation of Endocytotic Vesicles to Lysosomes
Requires Microtubules
After endocytosis, the ligand-containing
vesicles exhibit a saltatory movement at 30 nm/s, which is followed by
a rapid vectorial movement at 50 nm/s, until the vesicles fuse with
lysosomes (22) . In mammalian liver, the initial phase of
endocytosis does not require microtubules(23) . However, during
the vectorial translocation to lysosomes, endosomes remain associated
with microtubules(24, 25) . After the endosome is
acidified, the receptor dissociates from the ligand (26) and
the receptor-containing domain segregates from the ligand-containing
domain. The receptor-containing membranous domain returns to the cell
surface, whereas the ligand-containing vesicle translocates to the
lysosome, where its contents are degraded. Movement of the
ligand-containing vesicle to the lysosome along microtubules may be
powered by the ATP-utilizing motor, cytoplasmic dynein(25) .
The receptor-containing domain returns to the cell surface also along
microtubules, probably powered by the other motor molecules, such as
kinesin. Depolymerization of microtubules by colchicine treatment
causes redistribution of ASGR from the cell surface to the cytoplasm (23) . The directionality of movement of vesicles along
microtubules may depend on activation of specific groups of motor
proteins by phosphorylation of proteins that are associated with the
motor proteins (17) . Based on the above considerations it may
be predicted that microtubular disruption should interfere with the
later phases of receptor-mediated endocytotic pathway by inhibiting
receptor-ligand segregation (23, 24) and progression
of endosomes to lysosomes.
Persistence of Endocytosed DNA Depends on Early Events
Following Endocytosis
We have shown previously that normally
most of the DNA internalized by hepatocytes via ASGR is degraded during
the first 4 h after endocytosis(6) . Therefore, any maneuver
that results in persistence of a significant fraction of the
internalized DNA must be effective during the first few hours after DNA
administration. Partial hepatectomy results in persistence of
endocytosed genes in cytoplasmic vesicles of hepatocytes and prolongs
their expression(7) . The time course of degradation of the
endocytosed DNA indicates that such persistence cannot be the result of
DNA synthesis, karyokinesis, and mitosis of hepatocytes that occur
16-24 h after partial hepatectomy (7) . Therefore, the
protective effect of 66% hepatectomy on the internalized DNA appears to
result from events that occur within the first few hours after partial
hepatectomy. In previous studies, we have observed a significant degree
of disruption of the microtubular network 2-6 h after 66%
hepatectomy. This event is rapid enough to affect translocation of
endosomes to lysosomes, thereby inhibiting the degradation of endosomal
contents, which may explain the observed transgene
persistence(8) . To directly test the hypothesis that
microtubular disruption should result in persistence of the endocytosed
DNA, in this study we used colchicine pretreatment to disrupt
hepatocellular microtubules. Our finding that following microtubular
disruption the endocytosed DNA persists in the cytoplasm is consistent
with the postulate that during the period required for regeneration of
the disrupted microtubules, the ``derailed'' endosomal
vesicles containing the internalized DNA may undergo changes that
interfere with their association with microtubules or functional motor
proteins.
Optimum Time of Colchicine Injection in Relation to the
DNA Delivery
Because microtubule-disrupting agents interfere
with the return of ASGR to the plasma membrane, the receptor is
redistributed from the cell surface to the cytoplasm. Over a period of
time, this leads to depletion of cell surface receptors(23) ,
thereby reducing endocytosis. Therefore, initial experiments were
conducted to determine the time course of microtubular depolymerization
after colchicine administration to identify the correct time of
colchicine administration in relation to DNA delivery. These
experiments showed that injection of colchicine up to 30 min before
ASGR-mediated DNA delivery did not significantly reduce internalization
of the plasmid by the liver. Because microtubules were markedly
disrupted within 2 h after colchicine administration, this allowed the
opportunity to study the effect of microtubule disruption on the
persistence of the endocytosed plasmid.
Significance for Liver-directed Gene
Therapy
Receptor-mediated liver-targeted delivery of therapeutic
genes in vivo is an attractive method for gene therapy for
inherited diseases. However, attempts to use this method for treating
metabolic liver diseases, such as hyperlipidemia in low density
lipoprotein receptor-deficient Watanabe heritable hyperlipidemic
rabbits resulted in only short term effects(4) . Prolonged
persistence of the transgene has heretofore required partial
hepatectomy after gene delivery(5) , which obviously limits the
potential clinical applicability of this method. Dramatic prolongation
of transgene expression by pharmacological disruption of microtubules
provides a novel noninvasive technique for achieving transgene
persistence. The dose of chochicine used in this study would be
considered toxic in humans. However, other microtubule-disrupting
agents, such as Vinca alkaloids, may be more clinically
appropriate. Alternatively, receptor-mediated delivery of colchicine to
the liver may be used to limit its systemic toxicity.