help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2003-0366
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
145/1/261    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yoshida, M.
Right arrow Articles by Oiso, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yoshida, M.
Right arrow Articles by Oiso, Y.
Endocrinology Vol. 145, No. 1 261-268
Copyright © 2004 by The Endocrine Society

Gene Therapy for Central Diabetes Insipidus: Effective Antidiuresis by Muscle-Targeted Gene Transfer

Masanori Yoshida, Yasumasa Iwasaki, Masato Asai, Takeshi Nigawara and Yutaka Oiso

Departments of Medicine (M.Y., M.A., Y.O.) and Clinical Pathophysiology (Y.I., T.N.), Nagoya University Graduate School of Medicine and Hospital, Nagoya 466-8550, Japan

Address all correspondence and requests for reprints to: Yasumasa Iwasaki, M.D., Ph.D, Department of Clinical Pathophysiology, Nagoya University Graduate School of Medicine and Hospital, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: iwasakiy{at}med.nagoya-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Central diabetes insipidus, characterized by severe polyuria and polydipsia, is a disorder resulting from deficient secretion of the small neuropeptide hormone vasopressin in the neurohypophysis. The standard therapy is daily and life-long administration of vasopressin analog (desmopressin acetate), but gene therapy is potentially alternative to the conventional replacement therapy. To obtain the therapeutic neuropeptide more feasibly, we tried to express vasopressin in nonneuronal tissues using nonviral gene transfer techniques. We found that the unprocessed large precursor form, provasopressin, was predominantly produced in nonendocrine cells transfected with the wild-type vasopressin gene, because of the lack of neuroendocrine cell-specific endopeptidases. In sharp contrast, appropriately processed bioactive vasopressin can be efficiently produced even in nonendocrine cells with a modified vasopressin gene containing a ubiquitous endoprotease furin cleavage site. We also succeeded in maintaining a long-term antidiuretic effect on vasopressin-deficient (Brattleboro) rats by direct introduction of the furin-processible gene into skeletal muscle by electroporation. Altogether, our data clearly show that skeletal muscle is a useful target tissue for continuous delivery of bioactive neuropeptide. Furthermore, our strategies may be applicable to future gene therapies for central diabetes insipidus and other peptide hormone deficiencies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A SMALL PEPTIDE hormone arginine vasopressin (vasopressin), also known as antidiuretic hormone, is secreted from the neurohypophysis and plays a pivotal role in osmoregulation and water metabolism (1). Deficient vasopressin secretion results in central diabetes insipidus (CDI) with marked polyuria at an amount of more than 10 liters/d. Gene therapy with sustained expression of vasopressin may achieve long-term remission of this disease and can be used instead of the daily repeated replacement of vasopressin analog.

In contrast to GH or other protein deficiencies (2, 3, 4, 5), there are very few reports on gene therapy for CDI. Geddes et al. (6, 7) carried out a pioneering work, showing that the direct injection of adenovirus encoding vasopressin cDNA into the supraoptic nuclei of the hypothalamus of Brattleboro rats, an animal model of hereditary CDI, improved polyuria. This strategy, however, cannot be applicable clinically, because, unlike the Brattleboro rat in which vasopressin-secreting neurons are exceptionally preserved (8, 9), the homologous neurons are lost or degenerated in patients with CDI (10, 11). In addition, the induction of viral vectors into the human central nervous system does not appear safe or practical. Our aim of this work is to obtain bioactive vasopressin from nonneuronal tissues using a simpler and more feasible approach. When the vasopressin gene is to be introduced into heterologous cells, on the other hand, we face another obstacle, namely, that the processing of vasopressin peptide is deficient in nonendocrine cells. Like many other neuropeptide hormones, the bioactive nonapeptide form of vasopressin is generated from a large precursor, provasopressin, at the paired basic amino acid residues (-11Lys-12Arg-) by specific endopeptidase, such as prohormone convertase (PC) 1/3 or PC2, yielding neurophysin II which is a vasopressin-binding protein, and glycoprotein (12, 13, 14, 15) (Fig. 1AGo). Indeed, the vasopressin precursor protein expressed in nonendocrine cells is not appropriately processed (16), probably because of the lack of a processing enzyme(s).



View larger version (37K):
[in this window]
[in a new window]
  		FIG. 1. Processing of vasopressin precursor protein, preprovasopressin, with the associated processing enzymes. A, A scheme representing the posttranslational processing of vasopressin (AVP). After removal of a signal peptide (SP), provasopressin is further processed into three small peptides, vasopressin, neurophysin II, and C-terminal glycopeptide. Processing between vasopressin and neurophysin II is known to occur at the paired basic amino acid residues (-11Lys-12Arg-) by specific endopeptidases, resulting in a bioactive nonapeptide vasopressin. B, RT-PCR analyses of the endopeptidases PC1/3, PC2, and furin expression in a variety of cells/tissues used in this study. PC1/3, a neuroendocrine-specific endopeptidase, is expressed in GH3 and AtT20 but not in other nonendocrine cells/tissues. In contrast, a ubiquitous endopeptidase furin is expressed in all cell lines examined.

 
To overcome these limitations, we developed several strategies for expressing bioactive vasopressin even in heterologous nonendocrine cells. We succeeded in showing that mature-sized bioactive vasopressin can be produced efficiently in nonendocrine cells using mutated vasopressin cDNA, in which the amino acid sequences between vasopressin and neurophysin II were altered to be processed by a ubiquitous endoprotease, furin (17). In addition, we normalized the urine volume of the Brattleboro rats by direct im introduction of the furin-processible vasopressin gene using a potent nonviral in vivo gene delivery system, electroporation (18, 19, 20, 21), because skeletal muscle is an excellent target tissue for transgene expression (22, 23, 24). The achievement of constitutive processed neuropeptide secretion by muscle-targeted gene delivery paves the way for clinical application of gene therapies for CDI and various other hormone deficiencies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
RT-PCR
Expression of PC1/3, PC2, and furin mRNAs was analyzed by RT-PCR technique. Total RNA was extracted from each cell line using TRIzol reagent (Invitrogen, Carlsbad, CA), and RT-PCR was carried out using a commercially available kit (SuperScript, Invitrogen). The PCR primers used were: for mouse AtT20 cells, PC1/3, forward 5'-TTGGCTGAAAGGGAAAGAGAT-3', reverse 5'-ACTTCTTTGGTGATTGTTTTG-3'; PC2, forward 5'-GATCCTCTTTTTACAAAGCA-3', reverse 5'-AGCACTGTCAGATGTTGCAT-3'; furin, forward 5'-CCAGTTTTGACGTGAATGACC-3', reverse 5'-ATGGAGCCCAGCCCTCCTCG-3'; for rat GH3 and L6 cells, PC1/3, forward 5'-GTTGGCTGAAAGGGAAAGGGAT-3', reverse 5'-GAATCTTTGATGATTGCTTTGA-3'; PC2, forward 5'-GATCCTCTTTTTACAAAGCA-3', reverse 5'-AGCACTGTCAGATGTTGCAT-3'; furin, forward 5'-CCAGCTTTGATGTCAATGACC-3', reverse 5'-ATGGAGCCCAGCCCTCCGCG-3'; for COS1 and JEG3 cells, PC1/3, forward 5'-TTGGCTGAAAGAGAACGGAT-3', reverse 5'-ACTTCTTTGGTGATTGCTTTG-3'; PC2, forward 5'-GATCCTCTTTTTACAAAGCA-3', reverse 5'-AGCACAGTCAGATGCTGCAT-3'; furin, forward 5'-AAGTTTCCTCAGCAGTGGTA-3', reverse 5'-TTGTCATTCATCTGTGTGTACC-3'.

Plasmid construction
A wild-type (WT) vasopressin expression vector was constructed by inserting the rat vasopressin cDNA into pRc/RSV expression vector (CLONTECH, Palo Alto, CA). Furin-processible vasopressin expression vector (AVP/Fur) was made from WT by site-directed mutagenesis technique. CAG promoter-driven expression vector (pCAGGS/AVP/Fur) was constructed by inserting the AVP/Fur cDNA into pCAGGS expression vector (kindly provided by Prof. Miyazaki, Osaka University, Osaka, Japan) (25). PC1/3 and PC2 expression vectors were made by inserting the mouse PC1/3 or PC2 cDNA (kindly provided by Dr. Mains, Johns Hopkins University, Baltimore, MD) (26) into a pRc/CMV expression vector (CLONTECH).

Cell culture and transfection
AtT20, GH3, JEG3, COS1, L6, and LLC-PK1 cells were maintained with standard cell culture techniques. For transient expression, cells were plated in 35-mm-diameter culture dishes, and each vector was transfected using TransIT reagent (Mirus, Madison, WI). On the next day, the culture medium was changed to serum-free medium, and vasopressin secreted into the medium for 10 h was used for the subsequent analyses. Three separate dishes were used for each experimental condition. In most of the experiments, ß-galactosidase expression vector was used as an internal control. For stable transfection, L6 cells were transfected with pCAGGS/AVP/Fur using TransIT reagent. After selection with G418 (Geneticin, Invitrogen), clonal cell lines expressing vasopressin were selected, and the representative cell lines, designated as L6VP, were used for the subsequent experiments.

Gel filtration chromatography
Two milliliters of each culture medium from the transfected cells, or standard vasopressin (Peptide Institute, Osaka, Japan), was loaded and filtrated through a P-10 column (Bio-Rad, Hercules, CA) using a 4% acetic acid solution containing 0.1% BSA as a filtration buffer. Each fraction (8.0 ml each) was vacuum-dried, resuspended with assay buffer, and then applied for vasopressin RIA.

Vasopressin bioassay in vitro
LLC-PK1 cells were plated in 35-mm-diameter dishes and transfected transiently with cAMP response element (CRE x 5)-luciferase reporter gene using TransIT reagent. After 48 h, the cells were incubated for 5 h with serum-free medium containing standard vasopressin. The vasopressin bioactivity was determined by luciferase assay (27). Three separate dishes were used for each experimental condition. The same protocol was performed when the serum-free culture medium derived from vasopressin-expressing cells containing immunoreactive vasopressin (500 pg/ml, determined by RIA) with or without 1.0 µg/ml OPC31260 (provided by Otsuka Pharmaceutical Co., Tokyo, Japan) (28, 29), or control serum-free medium incubated with nontransfected cells was used. Each value obtained was normalized by the protein amount of the corresponding cell extract.

Animal experiments
Twenty-week-old male Brattleboro rats (kindly provided by Prof. Yamaoka, Dokkyo University, Tochigi, Japan) were used. All rats were kept under controlled lighting conditions (light, 0900 h; dark, 2100 h) and constant temperature (21 C). Animal surgery and care were in accordance with the Nagoya University institutional guidelines complying with the National Institutes of Health policy. For estimating in vivo bioactivity of vasopressin derived from the transfected cells in vitro, Brattleboro rats (n = 4 in each group) received a sc injection of culture medium (3 ml, 12 h incubation) derived from JEG3 or COS1 cells transiently transfected with AVP/Fur using LipofectAMINE PLUS (Invitrogen), or control medium incubated with nontransfected cells. Each rat was then placed in a metabolic cage for 2 h without water or food, and urine volume was determined by enforced urination every 30 min. For transplantation experiments, Brattleboro rats (n = 3 in each group) were sc transplanted with L6VP (1.2 x 108) cells with or without OPC31260. In the OPC-treated group, each rat received an ip injection of OPC31260 (10 mg/kg body weight, 1% in dimethylsulfoxide) on the d 4 or 6 after transplantation. As the control, Brattleboro rats (n = 3) transplanted with nontransfected L6 cells (1.2 x 108) were used. During these experiments, each recipient rat was housed in an individual metabolic cage, and daily urine volume and water intake, normalized by body weight, were determined. Food and water were available ad libitum. Urine osmolality was measured by OSMOSTAT OM6040 (ARKRAY, Shiga, Japan).

Electric pulse delivery and electrodes
Electric pulses were delivered using an electric pulse generator (Square Electroporator CUY 21 EDIT; NEPA GENE Co. Ltd., Ichikawa, Japan). Brattleboro rats (n = 3–4 in each group) were anesthetized with diethylether, and an approximately 5-cm incision was made in the skin of the left hind limb. pCAGGS-AVP/Fur or control vector (pCAGGS) (60 µg/site, 1 mg/ml in saline) was injected into the left anterior tibial and soleal muscles with a 27-gauge needle centered between a pair of stainless electrode needles (5 mm in length and 0.4 mm in diameter, with a fixed distance of 5 mm between them). Total plasmid amount per rat was 5 µg/g body weight. Immediately after plasmid injection, four pulses of 100 V, 50 msec, followed by four more pulses of the opposite polarity, were administered to each injection site at a rate of 1 pulse/sec. Each rat was then housed in an individual metabolic cage, with food and water available ad libitum. Daily urine volume and water intake were determined, and were normalized by body weight. Expression of vasopressin mRNA in injected muscle was analyzed by RT-PCR after deoxyribonuclease treatment. The PCR primers used were: forward 5'-GCCAGGAGGAGAACTACCTG-3', reverse 5'-ACCAGCCTAAGCAGCAGCTC-3'.

Vasopressin RIA
For the determination of tissue vasopressin, the electroporated muscle was homogenized with 0.1 N HCl and centrifuged, and the suprernate was extracted by Sep-Pak C18 column (Waters Associates Inc., Milford, MA) (30). For plasma vasopressin, rats were decapitated and trunk blood was collected in chilled tubes containing EDTA-2K. After immediate centrifugation, plasma was separated and vasopressin was extracted as mentioned above. Vasopressin was determined by a high-sensitive RIA kit (Mitsubishi Chemical Co., Tokyo, Japan) (31). The sensitivity of assay for arginine vasopressin was 0.063 pg/tube, with less than 0.01% cross-reactivity with oxytocin or lysine vasopressin.

Statistical analysis
All of the experiments were carried out more than twice to confirm the reproducibility, and the representative data are presented. Data are expressed as means ± SEM. Statistical comparison between the groups was made by one-way ANOVA with Fisher’s multiple range test. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Processing profiles of vasopressin expressed in endocrine or nonendocrine cells
First, we found by RT-PCR that the putative enzyme(s) for vasopressin processing, PC 1/3 and/or PC2, are expressed in GH3 rat somatomammotroph and AtT20 mouse corticotroph cells but not in COS1, JEG3, and L6 cells (Fig. 1BGo). When WT rat vasopressin cDNA expression vector was introduced, fully processed immunoreactive vasopressin, analyzed by gel filtration chromatography, was released from AtT20 cells (Fig. 2Go, A and B). In contrast, when WT expression vector was introduced into JEG3 and COS1 cells, vasopressin was not appropriately processed (Fig. 2Go, C, D, G, and H). In these cell lines, however, when PC1/3 expression vector was coexpressed, the processing profile was dramatically improved and vasopressin was secreted more efficiently (Fig. 2Go, C, E, G, and I). Coexpression of PC2 was not effective (Fig. 2Go, C, F, G, and J). Thus, simultaneous expression of peptide hormone and its specific processing enzyme is one of the methods for producing appropriately processed final products. Our data also suggest that PC1/3, but not PC2, is the enzyme responsible for the processing that occurs between vasopressin and neurophysin II.



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 2. Processing profiles of vasopressin expressed in endocrine (AtT20) or nonendocrine (JEG3, COS1) cells. A and B, Immunoreactive vasopressin (iAVP) in culture medium obtained from transfected AtT20 cells (A). The molecular size was analyzed by gel filtration chromatography (B). C–F, iAVP in culture medium in JEG3 cells transiently transfected with vehicle, WT alone, WT + PC1/3 coexpression, and WT + PC2 coexpression (C). The molecular size of iAVP in each culture medium is indicated. WT alone (D), WT + PC1/3 (E), and WT + PC2 (F). G–J, A similar experiment in COS1 cells. iAVP in culture medium (G). The molecular size of iAVP in each culture medium is indicated. WT alone (H), WT + PC1/3 (I), and WT + PC2 (J). Data are mean ± SEM (n = 3 in each group). *, P < 0.05 vs. control. Open or filled arrowheads represent void or total volume, respectively. Arrows indicate the peak of elution position of standard vasopressin using the same conditions.

 
Processing profiles of vasopressin transfected with furin-processible provasopressin gene
We also tried an alternative approach using modified provasopressin cDNA, in which the vasopressin and neurophysin II junction was altered to a tetra basic furin cleavage site (Fig. 3AGo). When the modified expression vector (AVP/Fur) was introduced into JEG3 or COS1 cells that express furin (Fig. 1BGo), a more efficient vasopressin secretion with a markedly improved processing profile was obtained (Fig. 3Go, B–E), compared with WT (Fig. 2Go, D and H).



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 3. Processing profiles of vasopressin derived from furin-processible vasopressin precursor protein. A, Scheme of the nucleotide sequences of the vasopressin-neurophysin II connection site in WT, or AVP/Fur vasopressin cDNA with two arginine insertions. B and C, Immunoreactive vasopressin (iAVP) in the culture medium of JEG3 cells transiently transfected with WT or AVP/Fur. The molecular size of iAVP in culture medium of AVP/Fur transfected JEG3 cells (compare with Fig. 2Go, C and D) is indicated. D and E, A similar experiment in COS1 cells. iAVP in culture medium (D). The molecular size of iAVP in culture medium of AVP/Fur-transfected COS1 cells is indicated (E) (compare with Fig. 2Go, G and H). Data are mean ± SEM (n = 3 in each group). *, P < 0.05 vs. WT transfection. Open or filled arrowheads represent void or total volume, respectively. Arrows indicate the peak of elution position of standard vasopressin.

 
Assessment of vasopressin bioactivity in vitro and in vivo
The bioactivity of secreted vasopressin in both strategies was confirmed using both in vitro and in vivo bioassay systems. LLC-PK1, renal epithelial cells known to express V2 vasopressin receptor (32), were treated with culture medium containing expressed vasopressin, and the bioactivity was monitored by cAMP/protein kinase A-stimulated CRE-luciferase reporter gene expression (Fig. 4AGo). The efficacy of this system was validated by standard vasopressin peptide (Fig. 4BGo). Vasopressin derived from WT or AVP/Fur in all cell lines stimulated CRE-luciferase activity, compared with the nontransfected control medium. Notably, AVP/Fur-derived vasopressin showed higher bioactivity than vasopressin derived from WT in JEG3 or COS1 cells, probably because of the improved processing efficiency (Fig. 4Go, D and E). We confirmed that these enhancements were completely inhibited after the treatment with the selective V2 receptor antagonist OPC31260 (Fig. 4Go, F and G), indicating that the secreted vasopressin increased the CRE-luciferase activity via the V2 receptor. Furthermore, when the AVP/Fur- derived culture medium was sc injected into Brattleboro CDI rats, the cumulated urine volume was markedly suppressed (Fig. 5Go). Collectively, these data indicate that AVP/Fur- derived immunoreactive vasopressin expressed in nonendocrine cells is physiologically bioactive both in vitro and in vivo.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 4. Assessment of vasopressin bioactivity in vitro. A, Scheme of the in vitro vasopressin bioassay system using LLC-PK1 cells expressing the intrinsic vasopressin V2 receptor. The degree of receptor stimulation is estimated by the CRE (x5)-driven luciferase activity instead of directly measuring cAMP production. B, Dose-response effect of standard vasopressin (100, 200, 500 pg/ml) on the above system. C–E, The bioactivity of vasopressin secreted from the WT or AVP/Fur transfected cells in vitro. LLC-PK1 cells transfected with CRE-luciferase reporter gene were treated with the test medium containing 500 pg/ml of immunoreactive vasopressin (iAVP) that were derived from WT or AVP/Fur transfected cells, or control medium (C; medium from nontransfected cells) for 5 h. AtT20 transfected with WT vector (C); JEG3 cells transfected with either WT or AVP/Fur vector (D); COS1 cells transfected with either WT or AVP/Fur vector (E). F and G, The effect of selective V2 receptor antagonist, OPC31260, on the above vasopressin bioassay system. LLC-PK1 cells expressing CRE-luciferase reporter gene were treated with control medium or test medium (containing 500 pg/ml of iAVP derived from JEG3 transfected with AVP/Fur) with or without 1.0 µg/ml OPC31260 (F). A similar experiment in COS1 cells (G). Data are mean ± SEM (n = 3 in each group). *, P < 0.05 vs. control.

 


View larger version (18K):
[in this window]
[in a new window]
  		FIG. 5. The bioactivity of vasopressin secreted from the transfected cells in vivo. The cumulated urine volume was estimated in Brattleboro rats injected sc with the test medium, derived from the AVP/Fur vector transfected JEG3 (A) or COS1 (B) cells (closed circles), or the control medium (derived from nontransfected JEG3 or COS1 cells) (open circles) (n = 4 in each group). The concentration of immunoreactive vasopressin was 8.2 ng/ml (JEG3) and 7.5 ng/ml (COS1). Data are mean ± SEM. *, P < 0.05 vs. control. Arrowheads represent the time of injection.

 
Antidiuretic effect of vasopressin-expressing L6 myoblasts
We then attempted to express vasopressin in live animals using nonviral in vivo gene delivery techniques. We selected skeletal muscle as the target organ to obtain bioactive vasopressin in vivo. To achieve higher levels of expression, we constructed a new expression vector, pCAGGS-AVP/Fur, in which AVP/Fur cDNA was driven by a more potent CAG promoter in myocytes (21, 25) (Fig. 6AGo). We confirmed that the expression vector indeed produced vasopressin more efficiently (>20-fold) than did RSV-AVP/Fur in the L6 rat skeletal muscle cell line (Fig. 6BGo). When L6VP cells in which pCAGGS-AVP/Fur were stably transfected were sc transplanted into Brattleboro rats, polyuria and polydipsia were significantly improved toward an almost normal range, accompanied with a marked increase of urine osmolality; in contrast, these parameters were not changed in L6 cell-transplanted control rats (Fig. 6Go, C–E). Moreover, the antidiuretic effect was blocked by the administration of OPC31260 for about 24 h, and this inhibition was reversed on the next day (Fig. 7Go), suggesting that the V2 receptor is responsible for the antidiuresis of L6VP-derived vasopressin, and the combination of V2 antagonists may prevent the water intoxication. Afterwards, this antidiuretic effect returned to the initial level without immunosuppressive drugs. However, these data indicate that myocyte-derived immunoreactive vasopressin exerts a potent antidiuretic effect via the V2 receptor as well as authentic vasopressin.



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 6. Antidiuretic effects of L6VP cell transplantation in Brattleboro rats. A, Scheme of pCAGGS/AVP/Fur vector. Furin-processible vasopressin cDNA is driven by CMV-IE enhancer and chicken ß-actin promoter. B, Immunoreactive vasopressin (iAVP) in the culture medium of L6 cells transiently transfected with pRSV/AVP/Fur or pCAGGS/AVP/Fur (n = 3 in each group). C and D, L6VP or L6 cells transplanted sc into the Brattleboro rats (n = 3 in each group). Daily urine volume (C) and water intake (D) were measured in both groups (data are shown as % of control L6-transplanted rats). E, Urine osmolality of Brattleboro rats 6 d after L6 or L6VP cell transplantation (n = 3 in each group). Data are mean ± SEM. *, P < 0.05 vs. control. Arrowheads represent the day of transplantation.

 


View larger version (20K):
[in this window]
[in a new window]
  		FIG. 7. The effect of OPC31260 on the antidiuresis induced by transplanted L6VP cells. On d 4 or 6 after L6VP transplantation, Brattleboro rats received OPC31260 administration. Daily urine volume (A) and water intake (B) were measured in both groups (n = 3 in each group). Data are shown as percentage of control L6-transplanted Brattleboro rats. Data are mean ± SEM. *, P < 0.05 vs. control. Arrowheads represent the day of transplantation.

 
Antidiuretic effect of vasopressin expressed in the skeletal muscle of Brattleboro rats
Finally, an electroporation technique was applied to deliver the vasopressin expression vector into skeletal muscles in vivo. After direct introduction of pCAGGS-AVP/Fur plasmid into unilateral (left) tibial and soleal muscles of Brattleboro rats by electroporation, the daily urine volume and water intake significantly decreased compared with control DI rats, to which control vector (pCAGGS) was introduced with electroporation (Fig. 8Go, A and B). Urine osmolality increased by up to 3-fold, compared with that of control (pCAGGS) rats. The antidiuretic effect was maintained for approximately 3 wk, and returned to the preinjection level on d 28. Food intake during the first 14 d after electroporation was similar in the pCAGGS-AVP/Fur and control (pCAGGS) rats (27.9 ± 0.5 vs. 25.9 ± 1.3 g/rat·d, respectively). Simple muscular injection of naked plasmid (pCAGGS-AVP/Fur) without electroporation failed to decrease urine volume (data not shown). Vasopressin expression in the muscle tissue receiving electroporation was confirmed by RT-PCR and protein analyses (Fig. 9Go, A and B). In the latter analysis, we observed substantial amounts of immunoreactive vasopressin in the muscle tissue 7 d after electroporation (384 ± 39 pg/g wet tissue), but not in the control (pCAGGS) rats (Fig. 9BGo). In addition, we observed a physiological concentration of plasma vasopressin in pCAGGS-AVP/Fur-introduced rats 7 d after electroporation (3.03 ± 0.66 pg/ml), compared with an almost undetectable level (0.06 ± 0.04 pg/ml) in the control rats (Fig. 9CGo). Collectively, we clearly demonstrated that nonendocrine cells such as myocytes can produce bioactive small peptide hormones like vasopressin in vivo.



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 8. Antidiuretic effect of vasopressin expressed in the skeletal muscle of Brattleboro rats. A and B, Direct introduction of pCAGGS/AVP/Fur or pCAGGS vector into the tibial and soleal muscles of Brattleboro rats by electroporation in vivo (n = 4 or 3, respectively). Daily urine volume (A) and water intake (B) were measured in both groups (open circles, data are shown as % of control pCAGGS electroporated rats). Data are mean ± SEM. *, P < 0.05 vs. control. Arrowheads represent the day of electroporation.

 


View larger version (20K):
[in this window]
[in a new window]
  		FIG. 9. A, Vasopressin mRNA expression in the electroporated muscle detected by RT-PCR. Amplified products from muscle tissues of DI rat (M), or from the hypothalamus of a normal Wister rat (H) as a control, are shown. B, Local immunoreactive vasopressin (iAVP) content of muscular tissue 7 d after electroporation of pCAGGS/AVP/Fur or control plasmid (pCAGGS). C, Plasma vasopressin concentration in pCAGGS/AVP-Fur or control plasmid (pCAGGS)-electroporated rats on d 7 (n = 4 and 3, respectively). Data are mean ± SEM. *, P < 0.05 vs. empty plasmid-introduced group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Gene therapies for supplying proteins to deficient patients are currently undergoing steady progress (33, 34, 35). In this paper, we tried to apply a nonviral gene transfer technique for the replacement therapy of small peptide hormones like arginine vasopressin. Our data clearly show that bioactive vasopressin can be produced efficiently even in nonendocrine cells both in vitro and in vivo so that fibroblast- or myocyte-derived vasopressin is enough to substitute for the degenerative vasopressinergic neurons of CDI patients. When the modified furin-processible gene was introduced into skeletal muscle of the hereditarily vasopressin-deficient Brattleboro rat, polyuria was markedly improved, promising the feasibility of these techniques to gene therapy for human CDI. Furthermore, the successful production of processed bioactive peptide hormones in nonendocrine cells/tissues is applicable to the replacement therapy of other peptide hormones.

The vasopressin gene encodes preprovasopressin, which consists of a signal peptide, neurophysin II, and glycoprotein, as well as vasopressin. After the removal of the signal peptide, provasopressin is further processed into the three end products by neuroendocrine cell-specific endoprotease PC1/3 or PC2. Both enzymes associate with the processing of proopiomelanocortin (36), proinsulin (37), and proglucagon (38). In nonendocrine cells without these enzymes, appropriately processed mature vasopressin is not produced, even though the vasopressin gene is efficiently expressed (16). We showed that coexpression of PC1/3, but not PC2, facilitated the processing between vasopressin and neurophysin II, resulting in improved secretion of immunoreactive vasopressin of an appropriate size even in nonendocrine cells. Alternatively, we constructed a modified vasopressin gene, the protein product of which is made processible by the ubiquitous endoprotease furin. This strategy is also effective, producing vasopressin much more efficiently than the WT gene. In this approach, the immunoreactive vasopressin is bioactive as well, as confirmed by both in vivo and in vitro assays. Thus, small peptide hormones like vasopressin can be produced in nonsecretory cells by either technique, i.e. coexpression of the target protein with its processing enzyme, or modification of hormone precursor genes into a processible form. The latter strategy has already been successfully applied for insulin gene expression (39, 40, 41). In addition, our data suggest that PC1/3 is the enzyme mainly responsible for the processing between vasopressin and neurophysin II. The mRNA of PC1/3 is known to colocalize with vasopressin mRNA in the magnocellular neurons of the hypothalamus (13).

Successful clinical applications of gene therapy rely on efficiencies in gene delivery, simplicity, safety, and duration of expression. The advantages of nonviral vectors over viral vectors are lower toxicity, lower immunogenicity, simplicity of use, and ease of large-scale production (34). Electroporation in vivo into skeletal muscle is more than 100-fold efficient compared with simple naked DNA injection (21), and successful gene delivery has been reported in a variety of cells and/or tissues (42, 43, 44). Moreover, skeletal muscle has the advantage of accessibility for gene delivery. The electroporation strategy in this study worked well and improved the polyuria of CDI rats for 3 wk. We performed electroporation with low-voltage and long-pulse currents, which is a highly efficient condition for gene transfer according to previous reports (21, 22). The duration of the effect of in vivo vasopressin expression was unexpectedly long, maintaining the improvement of polyuria for approximately 3 wk after a single electroporation procedure. Moreover, after the introduction of the expression vector, the plasma vasopressin concentration was within the physiological range, indicating that the myocyte-derived vasopressin can exert a similar bioactivity in vivo as does native vasopressin. Our data suggest that the expression of the processible form of vasopressin in skeletal muscle is a potential approach for long-term remission of CDI.

Implantation of L6VP cells exerted potent antidiuresis, but these cells were finally rejected with no immunosuppressive agents, suggesting that additional method(s) to prevent immunorejection would be necessary. Previous works have shown that the transplantation of bioengineered cells for supplying the desired gene products is a successful approach for gene therapy (2, 3, 45). For clinical application, the microencapsulation technique may be used for this purpose and also for preventing spreading of the transplanted cells (46). Alternatively, immunorejection may be avoided by use of host-derived myoblasts, fibroblasts, or even differentiated stem cells transfected ex vivo with therapeutic gene constructs and subsequent reimplantation (47, 48, 49, 50).

Finally, we have recognized that diabetes insipidus is a convenient model system for developing gene/cell therapy. Vasopressin is known to be effective for causing antidiuresis with very low plasma concentration (0.5–5 pM). The efficacy can be assessed easily, by the decrease in urine volume or the increase in urine osmolality, using the Brattleboro rat, or also probably the normal rat, if water loading is carried out. Furthermore, for clinical application, a relatively small amount of vasopressin is enough to decrease the daily urine volume to a convenient range. One remaining problem is that we cannot regulate the degree of plasma vasopressin when the vasopressin gene is constitutively expressed either in the transfected cells or in muscle. In the present study, we showed that the antidiuretic effect by expressed vasopressin was effectively inhibited by the selective vasopressin V2 receptor antagonist OPC31260, which is a nonpeptidic and orally effective agent (28, 29). Thus, the administration of V2 antagonists may be a useful approach to avoid water intoxication or regulate the urine volume under continuous vasopressin replacement. Overall, the strategies tested in this study using the DI rat will be helpful for future gene therapies for a variety of hormone deficiencies.


   Acknowledgments
 
We wish to thank Prof. Sadao Yamaoka for providing Brattleboro rats, and Prof. Jun-ichi Miyazaki for providing a pCAGGS expression vector. We also thank NEPA GENE Co., Ltd. for the Square Electroporator.


   Footnotes
 
This work was supported in part by a grant from Japanese Ministry of Health, Labor and Welfare (to Y.I.).

Abbreviations: AVP/Fur, Furin-processible vasopressin expression vector; CDI, central diabetes insipidus; CRE, cAMP-response element; WT, wild-type.

Received March 25, 2003.

Accepted for publication September 25, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Robertson GL 1976 The regulation of vasopressin function in health and disease. Recent Prog Horm Res 33:333–385
  2. Barr E, Leiden JM 1991 Systemic delivery of recombinant proteins by genetically modified myoblasts. Science 254:1507–1509[Abstract/Free Full Text]
  3. Dhawan J, Pan LC, Pavlath GK, Travis MA, Lanctot AM, Blau HM 1991 Systemic delivery of human growth hormone by injection of genetically engineered myoblasts. Science 254:1509–1512[Abstract/Free Full Text]
  4. Al-Hendy A, Hortelano G, Tannenbaum GS, Chang PL 1995 Correction of the growth defect in dwarf mice with non-autologous microencapsulated myoblasts—an alternate approach to somatic gene therapy. Hum Gene Ther 6:165–175[Medline]
  5. Rivera VM, Ye X, Courage NL, Sachar J, Cerasoli Jr F Wilson JM, Gilman M 1999 Long-term regulated expression of growth hormone in mice after intramuscular gene transfer. Proc Natl Acad Sci USA 96:8657–8662[Abstract/Free Full Text]
  6. Schmale H, Richter D 1984 Single base deletion in the vasopressin gene is the cause of diabetes insipidus in Brattleboro rats. Nature 308:705–709[CrossRef][Medline]
  7. Geddes BJ, Harding TC, Lightman SL, Uney JB 1997 Long-term gene therapy in the CNS: reversal of hypothalamic diabetes insipidus in the Brattleboro rat by using an adenovirus expressing arginine vasopressin. Nat Med 12:1402–1404
  8. Valtin H 1967 Hereditary hypothalamic diabetes insipidus in rats (Brattleboro strain). A useful experimental model. Am J Med 42:814–827[CrossRef][Medline]
  9. Majzoub JA, Pappey A, Burg R, Habener JF 1984 Vasopressin gene is expressed at low levels in the hypothalamus of the Brattleboro rat. Proc Natl Acad Sci USA 17:5296–5299
  10. Blotner H 1958 Primary or idiopathic diabetes insipidus: a system disease. Metabolism 7:191–200
  11. Bergeron C, Kovacs K, Ezrin C, Mizzen C 1991 Hereditary diabetes insipidus: an immunohistochemical study of the hypothalamus and pituitary gland. Acta Neuropathol (Berl) 3:345–348
  12. Ivell R, Richter D 1984 Structure and comparison of the oxytocin and vasopressin genes from rat. Proc Natl Acad Sci USA 81:2006–2010[Abstract/Free Full Text]
  13. Dong W, Seidel B, Marcinkiewicz M, Chretien M, Seidah NG, Day R 1997 Cellular localization of the prohormone convertases in the hypothalamic paraventricular and supraoptic nuclei: selective regulation of PC1 in corticotrophin-releasing hormone parvocellular neurons mediated by glucocorticoids. J Neurosci 17:563–575[Abstract/Free Full Text]
  14. Coates LC, Birch NP 1998 Differential cleavage of provasopressin by the major molecular forms of SPC3. J Neurochem 70:1670–1678[Medline]
  15. Zhou A, Webb G, Zhu X, Steiner DF 1999 Proteolytic processing in the secretory pathway. J Biol Chem 274:20745–20748[Free Full Text]
  16. Cwikel BJ, Habener JF 1987 Provasopressin-neurophysin II processing is cell-specific in heterologous cell lines expressing a metallothionein-vasopressin fusion gene. J Biol Chem 262:14235–14240[Abstract/Free Full Text]
  17. Nakayama K 1997 Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins. Biochem J 327:625–635
  18. Aihara H, Miyazaki J 1998 Gene transfer into muscle by electroporation in vivo. Nat Biotechnol 16:867–870[CrossRef][Medline]
  19. Mir LM, Bureau MF, Gehl J, Rangara R, Rouy D, Caillaud JM, Delaere P, Branellec D, Schwartz B, Scherman D 1999 High-efficiency gene transfer into skeletal muscle mediated by electric pulses. Proc Natl Acad Sci USA 96:4262–4267[Abstract/Free Full Text]
  20. Rizzuto G, Cappelletti M, Maione D, Savino R, Lazzaro D, Costa P, Mathiesen I, Cortese R, Ciliberto G, Laufer R, La Monica N, Fattori E 1999 Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation. Proc Natl Acad Sci USA 96:6417–6422[Abstract/Free Full Text]
  21. Somiari S, Glasspool-Malone J, Drabick JJ, Gilbert RA, Heller R, Jaroszeski MJ, Malone RW 2000 Theory and in vivo application of electroporative gene delivery. Mol Ther 2:178–187[CrossRef][Medline]
  22. Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, Felgner PL 1990 Direct gene transfer into mouse muscle in vivo. Science 247:1465–1468[Abstract/Free Full Text]
  23. Tokui M, Takei I, Tashiro F, Shimada A, Kasuga A, Ishii M, Ishii T, Takatsu K, Saruta T, Miyazaki J 1997 Intramuscular injection of expression plasmid DNA is an effective means of long-term systemic delivery of interleukin-5. Biochem Biophys Res Commun 233:527–531[CrossRef][Medline]
  24. Tsurumi Y, Takeshita S, Chen D, Kearney M, Rossow ST, Passeri J, Horowitz JR, Symes JF, Isner JM 1996 Direct intramuscular gene transfer of naked DNA encoding vascular endothelial growth factor augments collateral development and tissue perfusion. Circulation 94:3281–3290[Abstract/Free Full Text]
  25. Niwa H, Yamamura K, Miyazaki J 1991 Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108:193–199[CrossRef][Medline]
  26. Zhou A, Bloomquist BT, Mains RE 1993 The prohormone convertases PC1 and PC2 mediate distinct endoproteolytic cleavages in a strict temporal order during proopiomelanocortin biosynthetic processing. J Biol Chem 268:1763–1769[Abstract/Free Full Text]
  27. Iwasaki Y, Oiso Y, Saito H, Majzoub JA 1997 Positive and negative regulation of the rat vasopressin gene promoter. Endocrinology 138:5266–5274[Abstract/Free Full Text]
  28. Yamamura Y, Ogawa H, Yamashita H, Chihara T, Miyamoto H, Nakamura S, Onogawa T, Yamashita T, Hosokawa T, Mori T 1992 Characterization of a novel aquaretic agent, OPC-31260, as an orally effective, nonpeptide vasopressin V2 receptor antagonist. Br J Pharmacol 105:787–791[Medline]
  29. Fujisawa G, Ishikawa S, Tsuboi Y, Okada K, Saito T 1993 Therapeutic efficacy of non-peptide ADH antagonist OPC-31260 in SIADH rats. Kidney Int 44:19–23[Medline]
  30. Nagasaki H, Yokoi H, Arima H, Hirabayashi Y, Ishizaki S, Tachikawa K, Murase T, Miura Y, Oiso Y 2002 Overexpression of vasopressin in the rat transgenic for the metallothionein-vasopressin fusion gene. J Endocrinol 173:35–44[Abstract]
  31. Oiso Y, Iwasaki Y, Kondo K, Takatsuki K, Tomita A 1988 Effect of the opioid kappa-receptor agonist U50488H on the secretion of arginine vasopressin. Study on the mechanism of U50488H-induced diuresis. Neuroendocrinology 48:658–662[Medline]
  32. Ausiello DA, Hall DH, Dayer JM 1980 Modulation of cyclic AMP-dependent protein kinase by vasopressin and calcitonin in cultured porcine renal LLC-PK1 cells. Biochem J 186:773–780[Medline]
  33. Anderson WF 1998 Human gene therapy. Nature 392:25–30[CrossRef][Medline]
  34. Nishikawa M, Huang L 2001 Nonviral vectors in the new millennium: delivery barriers in gene transfer. Hum Gene Ther 12:861–870[CrossRef][Medline]
  35. Barzon L, Bonaguro R, Palu G, Boscaro M 2000 New perspectives for gene therapy in endocrinology. Eur J Endocrinol 143:447–466[Abstract]
  36. Bloomquist BT, Eipper BA, Mains RE 1991 Prohormone-converting enzymes: regulation and evaluation of function using antisense RNA. Mol Endocrinol 5:2014–2024[Abstract]
  37. Davidson HW, Rhodes CJ, Hutton JC 1988 Intraorganellar calcium and pH control proinsulin cleavage in the pancreatic ß cell via two distinct site-specific endopeptidases. Nature 333:93–96[CrossRef][Medline]
  38. Rothenberg ME, Eilertson CD, Klein K, Zhou Y, Lindberg I, McDonald JK, Mackin RB, Noe BD 1995 Processing of mouse proglucagon by recombinant prohormone convertase 1 and immunopurified prohormone convertase 2 in vitro. J Biol Chem 270:10136–10146[Abstract/Free Full Text]
  39. Yanagita M, Nakayama K, Takeuchi T 1992 Processing of mutated proinsulin with tetrabasic cleavage sites to bioactive insulin in the non-endocrine cell line, COS-7. FEBS Lett 311:55–59[CrossRef][Medline]
  40. Gros L, Riu E, Montoliu L, Ontiveros M, Lebrigand L, Bosch F 1999 Insulin production by engineered muscle cells. Hum Gene Ther 10:1207–1217[CrossRef][Medline]
  41. Lee HC, Kim SJ, Kim KS, Shin HC, Yoon JW 2000 Remission in models of type 1 diabetes by gene therapy using a single-chain insulin analogue. Nature 408:483–488[CrossRef][Medline]
  42. Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH 1982 Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1:841–845[Medline]
  43. Titomirov AV, Sukharev S, Kistanova E 1991 In vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNA. Biochim Biophys Acta 1088:131–134[Medline]
  44. Heller R, Jaroszeski M, Atkin A, Moradpour D, Gilbert R, Wands J, Nicolau C 1996 In vivo gene electroinjection and expression in rat liver. FEBS Lett 89:225–228
  45. Gage FH 1998 Cell therapy. Nature 392:18–24[Medline]
  46. Chang PL 1997 Microcapsules as bio-organs for somatic gene therapy. Ann NY Acad Sci 831:461–473[Abstract/Free Full Text]
  47. Campbell AI, Zhao Y, Sandhu R, Stewart DJ 2001 Cell-based gene transfer of vascular endothelial growth factor attenuates monocrotaline-induced pulmonary hypertension. Circulation 104:2242–2248[Abstract/Free Full Text]
  48. Suzuki K, Murtuza B, Smolenski RT, Sammut IA, Suzuki N, KanedaY, Yacoub MH 2001 Cell transplantation for the treatment of acute myocardial infarction using vascular endothelial growth factor-expressing skeletal myoblasts. Circulation 104:1207–1212
  49. Raymon HK, Thode S, Gage FH 1997 Application of ex vivo gene therapy in the treatment of Parkinson’s disease. Exp Neurol 144:82–91[CrossRef][Medline]
  50. Chen BF, Chang WC, Chen ST, Chen DS, Hwang LH 1995 Long-term expression of the biologically active growth hormone in genetically modified fibroblasts after implantation into a hypophysectomized rat. Hum Gene Ther 6:917–926[Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
145/1/261    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yoshida, M.
Right arrow Articles by Oiso, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yoshida, M.
Right arrow Articles by Oiso, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals