Department of 1Human Genetics and 2Medical Microbiology and Immunology, 3Medical Research Laboratories and Medical Department of Diabetes and Endocrinology, Aarhus University Hospital, DK-8000 Aarhus C, Denmark
Submitted 27 December 2002 ; accepted in final form 12 March 2003
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ABSTRACT |
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nonviral gene therapy; insulin-like growth factor I; insulin-like growth factor-binding protein-3; ubiquitin promotor; growth hormone deficiency
Considerable progress has been made in the development of stable and safe modes of transferring genes to somatic tissues. Although efficient in transferring foreign genes in vivo, the current virus-based systems have limitations. Vectors based on murine retrovirus cannot transduce nonproliferating cells, which makes it difficult to target cells with a low cell turnover. In addition, viral vectors have been associated with severe immune responses, making these unsafe and repeated administration complicated. Lentiviral vectors with the ability of transferring genes into quiescent cells deserve further investigations for liver gene therapy, although no clinical experience has been made yet.
Ex vivo techniques can be used to obtain circulating levels of exogenous proteins. In 1994, Heartlein et al. (7) were able to achieve stable and long-term (>500 days) levels of hGH in mice using modified autologous fibroblasts (transkaryotic implantation), but the levels obtained were relatively low (i.e., 14 µg/l), and no measurable effect on the body weight was observed. In 1995, al Hendy et al. (1) introduced microencapsulated myoblasts engineered to produce mouse growth hormone (mGH) in the Snell dwarf mice. The procedure resulted in an increased body size and growth rate for the initial 35 wk, but a significant difference between the mGH in treated and untreated mice could not be detected, and there was only a partial correction of the growth defect.
Systemic hydrodynamic (large volume injection) gene transfer is a promising new strategy to deliver and express foreign genes in vivo (2, 14, 20). Although the first reports showed a rapid decline in expression within days, new reports show that stable levels of gene expression after a single administration (2, 16) or after repeated injections (14, 19) can be achieved. The slow turnover of transfected hepatocytes in vivo in mature animals explains the long-term transgene expression, although the vectors are mainly present as nonintegrated episomes (2, 16). Modification of the hydrodynamic procedure using catheter-mediated delivery to the isolated liver may lead to a clinically practicable gene transfer method (4).
We have previously shown that hydrodynamic gene transfer of an hGH expression plasmid results in a high and sustainable expression of exogenous hGH in normal mice, despite the presence of anti-hGH antibodies (2). In this paper, we show that a single administration of a plasmid vector containing an hGH gene results in normalization of longitudinal growth and serum insulin-like growth factor I (IGF-I) in hypophysectomized mice.
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MATERIALS AND METHODS |
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Animals. The animal experiments were approved by the Danish Animal
Experiments Inspectorate. Male outbred mice (Bom:NMRI; M&B, Ry, Denmark),
8 wk old, were used for DNA injections. The animals were
hypophysectomized 3 wk before DNA injection by the transauricular method
(5). In brief, a modified
hypodermic needle was inserted through the auditory canal in the osseous bulla
on anesthetized animals. The pituitary gland was aspirated using a water
suction pump. The hypophysectomy was confirmed by a stunted body weight gain
during the following 3-wk postoperative period and shrinkage of the testis
compared with normal controls. Before hypophysectomy, the weight of each
animal was
26 g. After surgery (3 wk), the weight of each
hypophysectomized mouse was
23 g, whereas the normal mice each weighed
34 g. The mice were housed in plastic cages (Tecniplast, Buguggiate,
Italy) under pathogen-free conditions with a 12:12-h light-dark schedule and
fed standard chow (no. 1324; Altromin, Lage, Germany) and water ad libitum
throughout the experiment. At the end of the study, the animals were
anesthetized with pentobarbital sodium (4050 mg/kg mebumal; DAK
Nycomed, Roskilde, Denmark) for blood sampling and killed by cervical
dislocation before recovery.
Tail vein injections. Before the injection procedure, the animals
were kept at a high ambient temperature to dilate the tail veins. Anesthesia
was carried out in a chamber with 3.75% (vol/vol) isoflurane (Baxter Medical,
Kista, Sweden) in air until digital reflex was absent. Naked DNA was
administered to the animals by injection of 4550 µg plasmid DNA
contained in sterile Ringer solution (147 mM NaCl, 4 mM KCl, and 1.13 mM
CaCl2) in the tail vein. On the basis of data from previous studies
(14,
20), a volume of Ringer
solution corresponding to 8% of the body weight, i.e., 1.51.9 ml, was
used. Injection was performed within 39 s (average 4.9 s). After
injection, the weight of the mice increased according to the amount of volume
injected. After injection, the animals were allowed to recover with the
ambient temperature raised to 28°C. Hypophysectomized animals were
injected with the pUC-UBI-hGH construct (n = 9), with the pEGFP-N1
construct (n = 7), or left untreated (n = 7). Normal
(nonhypophysectomized) mice (n = 7) served as normal controls.
Blood sample and tissue collection. Blood samples were collected 7 days before plasmid injection and 13, 27, 42, 56, and 68 days after injection. Blood samples were drawn from the retroorbital plexus into vials (Microvette CB 300 KE; Sarstedt, Nümbrecht, Germany), and the serum was obtained subsequently by sample centrifugation at 1,000 g. At day 68, tissue samples were excised, weighed, and measured immediately after the animals were killed. Tissue samples for RNA extraction were frozen on dry ice and stored at -70°C. Analysis of green fluorescent protein (GFP) in liver samples mounted directly on slides and observed under a fluorescent microscope revealed high expression in mice injected with the pEGFP-N1 plasmid.
hGH and hGH antibodies. The concentration of hGH in serum samples
was measured by a commercial noncompetitive time-resolved immunofluorometric
hGH assay (TRIFMA; Delfia, Wallac, Finland) using two monoclonal antibodies
directed against different sites of the 22-kDa hGH variant. Serum samples were
examined for the presence of hGH antibodies by a precipitation method.
125I-labeled hGH [10,000 counts/min (cpm)] diluted in a 40% mmol/l
phosphate buffer (pH 8.0) was incubated with 25 µl serum at 4°C for 24
h. Precipitation was performed by addition of 100 µl newborn calf serum (to
facilitate -globulin precipitation) and 1.8 ml 20% polyethylene glycol
(PEG 6000) with 0.5% Tween 20 (both from Merck, Darmstadt, Germany) followed
by centrifugation at 3,600 rpm for 18 min. The presence of significant hGH
antibody formation was defined as a precipitation percentage larger than three
SDs of that obtained in control sera. This method relies on precipitation of
large proteins (molecular mass greater than
70 kDa) and is therefore not
specific for
-globulins. However, PEG has been used for decades in RIA
for precipitation of labeled antigens bound to specific antibodies
(3,
12).
Serum IGF-I. Serum IGF-I was measured after extraction with acid-ethanol. The extraction mixture was incubated for 2 h at room temperature followed by centrifugation, and 25 µl of the supernatant were subsequently diluted 1:200 before analysis. Serum IGF-I levels were measured by RIA using a polyclonal rabbit antibody (Nichols Institute Diagnostics, San Capristrano, CA) and recombinant human IGF-I as standard (Amersham International). Monoiodinated IGF-I (125I-labeled [Tyr31]IGF-I) was obtained from Novo-Nordisk (Bagsvaerd, Denmark). The intra- and interassay coefficients of variance were <5 and <10%, respectively.
Serum IGF-binding protein-3. SDS-PAGE and Western ligand blotting
were performed according to the method of Hossenlopp et al.
(8) as previously described
(6). Serum (2 µl) was
subjected to SDS-PAGE (10% polyacrylamide) under nonreducing conditions. The
electrophoresed proteins were transferred by electroelution on nitrocellulose
paper (Schleicher & Schuell, Munich, Germany), and membranes were
incubated overnight at 4°C with 500,000 cpm 125I-IGF-I (sp
act 2,000 Ci/mmol) in 10 ml 10 mmol/l Tris · HCl buffer containing 1%
BSA and 0.1% Tween (pH 7.4). Membranes were washed with Trish-buffered saline
and dried overnight, and the nitrocellulose sheets were subsequently
autoradiographed with Kodak X-AR film and exposed to DuPont-NEN (Boston, MA)
enhancing screens at -80°C for 37 days. Specificity of the
IGF-binding protein (IGFBP) bands was ensured by competitive coincubation with
unlabeled IGF-I purchased from Bachem (Bubendorf, Switzerland). On Western
ligand blotting (with 125I-IGF-I as ligand), IGFBP-3 appears as a
38- to 42-kDa doublet band corresponding to the intact acid-stable IGF-binding
subunit of IGFBP-3. Western ligand blots were quantified by densitometry using
a Shimadzu CS-9001 PC dual-wavelength flying spot scanner.
RNA extraction and RT-PCR. RNA samples were isolated from liver,
heart, lung, spleen, kidney, intestine, brain, and skin tissue from killed
mice using a High Pure RNA Tissue Kit (Roche, Mannheim, Germany). Samples were
collected 30 days after the hydrodynamic gene transfer. cDNA was prepared from
total RNA by (oligo)dT primed RT synthesis [First-Strand Synthesis Kit for
RT-PCR (AMV); Roche]. hGH cDNA was amplified by PCR using primers spanning a
187-bp region between the sense primer 5'-agcccgtgcagttcctcagg-3 and the
antisense primer 5'-gcaagttcgac-acaaactca-3'. A control for
template abundance was performed in parallel reactions using -actin
primers spanning a 196-bp region between sense primer
5'-CTGTGCTGTCCCTGTATGCC-3' and antisense primer
5'-GTGGTGGTGAAGCTGTAGCC-3'. PCR amplification with either set of
primers was carried out with a thermal program consisting of 30 cycles of
94°C for 30 s, 59°C for 30 s, and 74°C for 1 min.
Monitoring of growth. The tail length was measured with a calliper every week, and the body weight was measured two times a week. Measurement started 10 days before injection and continued throughout the study (68 days). At the time of death, the tibia was isolated, and the length was measured using a calliper. Selected organs (spleen, heart, lungs, and liver) were isolated and weighed.
Statistical analyses. A two-tailed Student's t-test was used for statistical analyses.
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RESULTS |
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Serum hGH and hGH antibodies. After administration of the
pUC-UBI-hGH plasmid, high levels of hGH protein could be detected in serum.
After an initial increase to 67.1 µg/l 13 days after injection, the
concentration remained stable at a level of 50 µg/l throughout the
study (Fig. 1A).
Interference of endogenous mGH was avoided by the use of an hGH specific
assay. As expected, hGH could not be detected in mice receiving pEGFP-N1,
untreated mice, or in the normal mice. Mice injected with the hGH plasmid
developed antibodies against hGH. In a blood sample taken 68 days after
injection, the hGH antibodies were detected using a method based on PEG
precipitation of radiolabeled hGH (data not shown). However, this had no
apparent impact on the effect on serum IGF-I, longitudinal growth, or increase
in body weight (see below).
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Serum IGF-I and IGFBP-3 levels. To investigate the physiological effect of the elevated hGH levels, we measured serum IGF-I, the major physiological mediator of GH actions. After an initial rise of IGF-I to above the levels found in normal mice (599 ± 33 compared with 373 ± 29 µg/l 13 days after DNA injection), serum IGF-I stabilized at a level comparable with that found in normal mice (Fig. 1B). From 40 days after treatment and onward, no significant difference in serum IGF-I between intact mice and mice treated with pUC-UBI-hGH plasmid was detected. As expected, the IGF-I levels were very low in mice receiving pEGFP-N1 and in uninjected mice. It is known that GH, in addition to stimulating IGF-I synthesis, stimulates the formation of ternary IGF-binding complexes, including IGFBP-3 (13). IGFBP-3 was measured in samples taken 7 days before treatment, and at death. A normalization in IGFBP-3 was observed in mice treated with pUC-UBI-hGH, whereas mice treated with pEGFP-N1 and untreated mice remained stable at the initial low level (Fig. 1C). Interestingly, IGFBP-3 also increased in the normal mice over time, probably because of a pubertal/postpubertal surge.
RNA analysis. To investigate plasmid DNA expression in various tissues, a subset of animals was killed and analyzed 27 days after injection. RNA was purified from tissues and subsequently used as template in RT-PCR reactions to examine the presence of hGH mRNA. After 27 days, hGH mRNA was detected exclusively in the liver (Fig. 1D), in agreement with previous findings (2, 16).
Body weight gain, longitudinal growth, and organ weight. Within
few days after plasmid injection, an increase in body weight and tail length
was observed in pUC-UBI-hGH plasmid-injected mice, whereas mice injected with
pEGFP-N1 plasmid and uninjected mice maintained a stable body weight and tail
length throughout the study period (Fig. 2,
A, B, and D). After a catch-up period of 3
wk for the body weight, and 5 wk for the tail length, the treated mice
stabilized at a growth rate similar to normal nonhypophysectomized mice. Tail
length was normalized and showed no significant difference in comparison with
normal mice, whereas body weight stabilized at a level of 45 g below
that of normal mice. A different measure for longitudinal growth, i.e., tibia
length, was determined at the end of the study. Tibia length was also
normalized, and no significant difference was found between mice treated with
pUC-UBI-hGH and normal mice. Mice injected with the pEGFP-N1 plasmid and
uninjected mice had significantly shorter tibia length
(Fig. 2C). The effect
of the hydrodynamic gene transfer on the weight of different organs was
assessed after 68 days, when all remaining mice were killed
(Fig. 3). In hypophysectomized
mice treated with pUC-UBI-hGH, the average weight of all investigated organs
was significantly higher than in the corresponding mice treated with pEGFP-N1
and untreated mice. Compared with normal mice, no significant difference in
organ weight was found for the liver, lungs, and spleen. However, the weight
of the heart was significantly higher in normal mice than in mice treated with
pUCUBI-hGH.
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DISCUSSION |
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Although hGH substitution in children and adult patients with GHD has been achieved successfully by daily subcutaneous injections of recombinant hGH (9), the search for alternatives to this treatment is still required. The GH levels in normal individuals are controlled through a complex interdependent secretion of hormonal factors, which is difficult to mimic by daily protein injections. Lifelong subcutaneous injections may be considered inconvenient by some patients with the risk of decreasing compliance (18). Furthermore, the cost of treatment by administration of rhGH is considerable, currently amounting to more than $10,000/patient annually. These concerns point toward in vivo synthesis by simple gene transfer as an appealing, potential alternative.
Using microencapsulated myoblasts, al Hendy et al. (1) obtained partial correction of the growth abnormalities seen in the Snell dwarf mice. The protein resulted in an increase in length and body weight and increased size of the tibial epiphyseal cartilage in the treated group. In the first 35 wk, an increase in growth rate was observed, and the myoblast continued to be actively secreting mGH throughout the study (178 days). However, as described above, correction of the growth abnormality was only partially normalized; furthermore, there was no detectable rise in circulating GH, and finally IGF-I was not measured (1).
Normal mice have pulsatile GH levels
(15). Baseline values are
2 µg/l, whereas peak values may reach 100 µg/l. In the present
study, hGH plasmid transcription and translation are independent of
endogenous mediators and are therefore expected to be relatively stable. We
found a concentration of 4050 µg/l, which is below the peak levels
in normal mice but
20-fold higher than the baseline levels. The effects
of constantly elevated hGH levels have to be investigated further, considering
that high GH and IGF-I levels may be associated with the development of
neoplasm (11). It is
intriguing, however, that, although GH levels in the injected mice were
20-fold higher than physiological baseline levels, the IGF-I and IGFBP-3
levels were normalized and comparable to levels in normal mice. The regulatory
mechanisms have to be investigated further, but these findings seem to
indicate that, to achieve adequate treatment, strict regulation of expression
is not necessary. Another possible explanation would be that hGH is less
efficient than mGH. Unlike GH, IGF-I is not under pulsatile secretion in
normal individuals, and serum IGF-I is normally relatively stable. Thus the
presumably stable IGF-I levels found in hGH-injected mice mirror the
physiological levels. We do not know why the hypophysectomized mice do not
catch up in body weight, although length and weight of most internal organs
are normalized. One explanation may be the known lipolytic effect of hGH,
although the lack of the other pituitary hormones in treated animals cannot be
ruled out.
Several groups have investigated the safety of the hydrodynamic gene transfer procedure, and no permanent damage on liver or other organs has been observed (14, 16, 19, 20). However, serious obstacles remain to be solved before the procedure can be used in a clinical setting. Hydrodynamic transfer has been performed successfully in larger animals [for example in dogs (21)] after open surgery, but ideally more convenient routes for gene transfer into humans are preferable. Access through the choledoctus duct or the cava vein may be advantageous since these vessels are relatively easy to reach through either endoscopic retrograde cholangiopancreatography or the femoral vein.
Although the current protocol cannot directly be applied in humans, the data strongly suggest that nonviral hGH gene transfer may be a feasible alternative to lifelong, daily hGH injections in GHD patients. However, further studies are warranted to fully unravel the potential use in humans.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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