- Email: [email protected]
DIRECT IN VIVO GENE TRANSFER TO UROLOGICAL ORGANS
0022-5347/99/1623-1115/0
Vol. 162, 111S1118,September 1999 Printed in U S A .
THE JOURNAL OF UROLOGY Copyright 0 1999 by AMERICAN UROLOCICAL ASSOCIATION, INC.
DIRECT IN VIVO GENE TRANSFER TO UROLOGICAL ORGANS JAMES J . YOO, SHAY SOKER, LEE F. LIN, KATHRYN MEHEGAN, PAUL D. GUTHRIE AND ANTHONY ATALA* From the Laboratory for Tissue Engineering and Cellular Therapeutics, Department of Urology, Children's Hospital and Harvard Medical School, Boston, Massachusetts
ABSTRACT
Purpose: Patients with urological disorders may benefit from gene based therapy. We investigated the feasibility of delivering exogenous genes into urological tissues in vivo using direct in vivo electrotransfection. Materials and Methods: Gene transfer to rat kidneys, testes and bladders was accomplished via direct local injection of pGL3/luciferase and P-galactosidase reporter gene constructs, followed by a n electrical pulse ranging from 55 to 115 msec. at 100 V. Direct injection of deoxyribonucleic acid without a n electrical pulse served as the control. The transfected and nontransfected organs were retrieved and analyzed by luciferase activity assay, histochemical and immunocytochemical staining for P-galactosidase, and reverse transcription polymerase chain reaction with primers specific for P-galactosidase messenger ribonucleic acid. Results: There was significant luciferase activity 1, 3 and 5 days after direct in vivo electrotransfection in kidneys and testes, and after 3, 5, 7 and 10 days in bladders. Positive P-galactosidase enzyme activity and P-galactosidase immunoreactivity were observed in the transfected renal tubular cells, testicular interstitial and germ cells, and uroepithelial bladder layer. Reverse transcription-polymerase chain reaction products of the transfected organs were noted, indicating the successful transcription of messenger ribonucleic acid. Conclusions: This study demonstrates that direct in vivo electrotransfection is a feasible method of transient gene delivery into intact urological organs. Its apparent safety and relative simplicity suggest that direct in vivo electrotransfection may be useful clinically. KEY WORDS:gene transfer, electroporation, kidney, testis, bladder
Recent advances in genetic engineering and molecular techniques have facilitated the production of various synthesized genes for therapeutic purposes. These technological advances have allowed the initiation of gene targeted therapies in various diseases Methods of in vivo gene based therapy are currently being approached by using viral or nonviral vectors. Gene transfer using viral vectors (retroviral and adenoviral) is achieved by infectivity of the viral particles. Although mammalian viral based vectors have gained wide acceptance, they have been associated with several problems. Retroviral vectors require replicating cells for integration and expression, and they carry only relatively small segments of deoxyribonucleic acid (DNA).4-6 Adenoviral vectors necessitate repeat administration due t o transient expression and they are associated with strong antigenecit^.",^ In contrast, nonviral gene transfer systems using naked plasmid DNA are considered safe and Unlike viral vectors the introduction of plasmid DNA into target cells is facilitated by chemical or physical Electroporation is a physical method of introducing macromolecules into cells in vitro by applying a brief electrical pulse that causes transient molecular membrane transit. This method provides higher transfer efficiency compared with other nonviral transformation methods."-" We developed a method to achieve organ confined gene transfer into urological organs in vivo, which we termed direct in vivo electrotransfection. Based on the principle of electroporation we designed a gene transfer device that would be effective in intact hollow and solid organs. This study was designed to determine the effectiveness of direct in vivo electrotransfection in intact kidneys, testes and bladders.
MATERIALS A N D METHODS
P l a s m i d s . Two vector systems, pGL3 and pCMVp, were
used for the transfection studies. pGL3 contains a reporter gene encoding firefly luciferase expressed by an SV40 promoter enhancer. pCMVp, driven by a cytomegalovirus promoter enhancer, contains the Escherichia coli lacZ gene encoding p-galactosidase. Plasmids were prepared by standard alkaline lysis techniques followed by ethanol precipitation. Phosphate buffered saline was used as the transfection buffer. A n i m a l model a n d gene transfer techniques. A total of 168 male Sprague-Dawley rats were studied, including the kidney, testis and bladder in 56 each. Gene transfer was performed with a device specifically designed for this study. The device was equipped with 2, 560 pF.1250 V. electrolytic capacitors charged in parallel through a limiting resistance to the desired voltage. The capacitors were subsequently disconnected from the power supply and discharged through the organ studied. Gene transfer to rat kidneys, testes and bladders was accomplished via direct local injection with a n 18 gauge silicone catheter. After the introduction of plasmid DNA into each kidney, testis and bladder organ contact was achieved with a negative electrode needle through the sheath. A positive electrode was placed externally. A current of 100 V. was applied to all organs. Mean treatment time plus or minus standard deviation was 89 -+ 6 msec. in kidneys, 105 2 10 in testes and 57 2 3 in bladders (range 55 to 115)depending on the specific tissue resistance. A dose of 100 pg. pGL3 was injected unilaterally into 42 kidneys and 42 testes, and instilled through a catheter into 42 bladders before the electric pulse was applied. Transfected * Requests for reprints: Children's Hospital and Harvard Medical and nontransfected organs (liver, spleen, kidney, testis and bladder) were retrieved on days 0, 1 , 3 , 5 , 7 , 10 and 14 after School, 300 Longwood Ave., Boston, Massachusetts 02115. 1115
1116
DIRECT IN VIVO GENE TRANSFER TO UROLOGICAL ORGANS
transfection for luciferase activity assay analysis (6per time point). Direct injection of DNA without a n electrical pulse served as the control. One organ transfection session was performed per animal. To determine the sites of cellular uptake and expression of the transfected genes kidneys, testes and bladders in another set of animals were transfected with pCMVp, an expression plasmid encoding the gene for E. coli p-galactosidase (lacZ) under the conditions described. Transfection with phosphate buffered saline only (no DNA) served as the control. The 14 testes, 14 kidneys and 14 bladders were retrieved at 1 , 3 and 5 days, respectively, for histochemical, immunocytochemical and reverse transcriptase (RT) polymerase chain reaction (PCR) studies. Luciferase activity assay. The 42 retrieved rat kidneys, 42 testes, 42 bladders and distant organs were frozen in liquid nitrogen. Each organ was homogenized in a tissue grinder and lysed in 1x lysis buffer (25 mM. tris, pH 7.8, with phosphoric acid, 2 mM. 1,2-diaminocyclohexe-N,N,N', tetraacetic acid, 2 mM. dithiothreitol, 10% glycerol and 1% Triton X-100) at room temperature for 15minutes. ARer centrifugation 20 pL. protein lysates were mixed with 100 pL. substrate, consisting of 270 pM. coenzyme A (lithium salt), 470 pM. luciferin and 530 pM. adenosinetriphosphatase. Luciferase activity was measured with a scintillation counter in triplicate in each organ. Statistical evaluation was performed using the Student t test with p 5 0.05 considered significant. Histochemical and imrnuruxytochernical studies. Retrieved organs were thoroughly flushed in phosphate buffered saline and frozen in OCT embedding compound. Cryostat sections (6 jm.were ) fixed in 4% paraformaldehyde for 10 minutes and subsequently stained with X-gal (5-bromo-4-chloro-3-indolyl-~ galahside). Fgalahsidase cleaves this substrate into an indigo compound, such that cells producing the transferred Pgalahsidase gene product are stained blue. Working solution for X-gal consisted of 10 mM. potassium ferricyanide, 10 mM. potassium ferrocyanide, 2 mM. magnesium chloride, 0.02% NF'-40,0.01% sodium deoxycholate and 0.4 mgJml. x-gal in dimethylformamide. Immunocytochemical study was performed with a mouse monoclonal anti-egalactosidase antibody using the avidin-biotin detection system. Subsequently tissue sections were counterstained with hematoxylin. RT-PCR for ribonucleic acid (RNA) analysis. RNA was extracted using a standard TRIzol method with some modifications." Each organ was homogenized in TRIzol reagent and RNA was precipitated with isopropyl alcohol. Subsequently RNA was mixed in a solution consisting of l x PCR buffer, 0.01 units per pl. ribonuclease inhibitor and 0.04 units per pl. deoxyribonuclease I. The reaction mixture was incubated at 37C for 30 minutes. Immediately after incubation 0.25 pg./pl. proteinase K was added to the reaction mixture and incubated again for 30 minutes. After precipi-
tation of RNA complementary DNA synthesis was performed using oligo d(T) primer, as described by the manufacturer. PCR was performed using a n amplification cycle profile consisting of 94C for 1 minute, 62C for 1 minute and 72C for 2 minutes per cycle. After 30 PCR cycles a n additional cycle at 72C for 7 minutes was performed to ensure complete DNA extension. RESULTS
All animals survived without any complications. At retrieval the transfected and distant organs appeared normal grossly and histologically. Luciferase activity in the transfected kidneys was measured (6 per time point). Peak luciferase activity was noted at days 3 and 5 in the electroporated kidneys (fig. 1, A ) . Kidneys injected with DNA without electroporation also showed luciferase activity, although reporter activity was consistently weaker than that in electroporated kidneys (p <0.05). In the transfected testes significant luciferase activity was expressed 1, 3 and 5 days after electrotransfection, and lower levels of activity were detected throughout the remaining course of the study (6 specimens per time point, p <0.05, fig. 1,B). Luciferase activity in the control testes injected with DNA without electrical pulse was not significant. In contrast, the 42 bladders consistently had maximal expression at day 7 (fig. 1, C). Bladders instilled with DNA only as the controls failed to show any activity. Nontransfected organs retrieved from electrically pulsed animals 0, 1,3, 5 7, 10 and 14 days after the procedure consistently failed to demonstrate any luciferase activity greater than the background level. Histochemical and immunocytochemical analyses of P-galactosidase expression in the transfected tissue revealed similar results. In the 8 kidneys positive staining was confined to the renal cortex, while the medulla failed t o stain. Renal tubular cells stained positively but other structures, such as glomeruli, collecting ducts and interstitial cells, did not stain (fig. 2, A and B). In the 8 transfected testes there was positive 6-galactosidase expression in interstitial and germ cells in the lumen of the seminiferous tubules (fig. 2, C and D).In the 8 bladders at day 7 the uroepithelial cell layer stained positive. However, the submucosal and smooth muscle layers failed t o stain (fig. 2, E ) . None of the phosphate buffered saline treated control tissues or distant organs not transfected revealed any P-galactosidase activity. As an independent assay for gene expression, RT-PCR was performed. RT-PCR products of expected size (500 bp) were obtained with RNA from 6 kidneys, 6 testes and 6 bladders transfected with pCMVj3. Control tissues failed to show any p-galactosidase messenger (m) RNA by RT-PCR. These findings indicate that P-galactosidase mRNA was transcribed in the transfected kidneys, testes and bladders (fig. 3).
FIG. 1. Time course of luciferase gene expression after direct in viva electrotransfection (DIVE)with 100 pg. pGL3. A, peak activity in kidneys at days 3 and 5. B , significant activity in testes a t days 1, 3 and 5. C , peak activity in transfected bladders at day 7.
DIRECT IN VIVO GENE TRANSFER TO UROLOGICAL ORGANS
1117
FIG. 2. Histochemical and immunocytochemical analyses of P-galactosidase expression. A, indigo blue staining in renal cortex but not in medulla in transfected kidneys. X-gal staining, reduced from X25. B . 6-galactosidase expression only in renal tubular cells with glomeruli and stromal cells staining negatively. X-gal staining, reduced from X250. C , indigo blue staining in interstitium and germ cells in direct in vivo electrotransfection testis. X-gal staining, reduced from X25. D , immunocytochemical study using monoclonal P-galactosidase antibody shows positively stained germ cells in seminiferous tubule. Reduced from X400. E , immunocytochemical analysis of P-galactosidase expression using p-galactosidase monoclonal antibody in transfected bladders with positive staining over whole urothelial cell layer. Reduced from X100.
1
2
3
4
5
6
7
bP
--600 ---
700
500
---
400
---
FIG. 3. RT-PCR products indicate P-galactosidase mRNA in transfected bladders (2), kidneys ( 4 ) and testes ( 6 ) .Control bladders (3), kidneys (5)and testes (7) failed to show a n y P-galactosidase mRNA. 1 , positive
control p-galactosidase plasmid. DISCUSS I 0 N
Urological disorders of the kidney, testis and bladder may be treated by introducing specific genes in a manner that may correct the malfunctioning cellular components. In this study we explored the possibility of delivering exogenous genes to intact urological organs. For the gene transfer methods we used the principle of electroporation. Electroporation is an established in vitro method of gene transfer in cultured cells with relatively high efficiency.", l1 Recently gene transfer by electroporation has been attempted in several tissues, including the muscle, skin and vessel^.'^-^^ I n this study we designed an electroporation gene transfer device for targeting whole urological organs in vivo. Based on the fact that electrical current flows between electrodes we designed the system to transmit mild electrical pulses to the target organ. Transmission was made possible by discharging electrical current through a central electrode needle toward the other node, thus, encasing the whole organ externally. This technique enabled the current to travel omnidirectionally from the centrally located electrode toward the surface of the target organ. The applied electrical field transmitted by the
direct in vivo electrotransfection device affects the whole organ. This system is an improvement over the in vivo electroporation system used in previous studies, in which the range of electrical current was limited by the area inside the e1e~trodes.l~~~ Histological examination of the electrotransfected organs revealed no evidence of damage subsequent to transfection and there were no apparent systemic or distant organ effects. Various therapeutic modalities involving electrical sources have been applied for clinically managing certain diseases in humans, including cutaneous electrostimulation for pain relief and electroconvulsive therapy in manic depressive disorders. These widely accepted therapeutic applications indicate that controlled exposure of a patient to electrical current is safe. We demonstrated in thisstudy that a current setting of 100 V. is efficient for gene transfer based on the quantitation of reporter gene activity and is within the current settings used in other medical modalities (range 50 to 130 V.). Most importantly current exposure time is limited to msec., providing a virtually painless and rapid procedure. Gene transfer systems using naked plasmid DNA vectors usually result in transient e x p r e ~ s i o n . ~In - ~ our current study using pGL3 there was also a limited duration of gene expression. However, the use of nonintegrating episomal expression vectors that replicate extrachromosomally may allow this procedure to be applied when prolonged expression of the transferred genes is desirable. Safety has been a continued concern in the development of gene therapy protocols. Although a number of clinical trials of various vector systems have been satisfactory, the possibilities of viral infection, mutagenesis and antigenecity may be further decreased by using naked plasmid DNA.We demonstrated that expression of genes may be elicited from nonindependently replicating plasmid DNA, indicating that commonly used recombinant expression vectors may be used in this procedure. Plasmids may also be purified to homogeneity by straightforward techniques, decreasing the risk of immunological reaction to foreign proteins. This latter feature should also allow repeat treatments without the risk of immunological complications. The kidney is an attractive target organ for gene delivery because it may be manipulated by percutaneous methods.16
1118
DIRECT IN VIVO GENE TRANSFER TO UROLOGICAL ORGANS
The direct injection of plasmids resulted in reporter gene expression. This result is consistent with those in previous reports of successful gene transfer in vivo by direct injection of naked DNA into tissues, such as the heart, liver and skeletal muscle. 17-19 Nevertheless, renal gene transfer was enhanced by using direct in vivo electrotransfection. Analysis of Pgalactosidase expression by in situ enzyme assay and immunocytochemical study aRer transfection with a lacZ reporter plasmid indicated that renal tubular cells expressed the transfected gene while other structures, such as glomeruli, collecting ducts and interstitial cells, did not express the gene or expressed it at a lower level. These results resemble those of Moullier et al, who delivered a replication defective adenovirus carrying a lacZ reporter cassette to the kidney by direct infusion into the renal artery.20 P-galactosidase expression was observed in the tubular epithelial cells of the renal cortex with no evidence of gene transfer in glomerular structures, or vascular or interstitial compartments. Although to our knowledge the mechanisms underlying these apparent differences in cell targeting are unknown, these results suggest that tubular epithelial cells, which comprise greater than 90% of the constituent cells of the kidney, may be preferential targets for gene therapy using the direct in vivo electrotransfection method. Our study shows the possibility of introducing foreign genes in testes. P-galactosidase gene expression was detected in various cell types, including interstitial and germ cells. Specific genes regulating tumor cell suppression or killing may be directly delivered t o affected cells using direct in vivo electrotransfection. In the future testicular gene transfer may lead to the production of transgenic animals and may be applied as a treatment strategy for hereditary disease. Gene transfer to bladders using electrotransfection resulted in gene delivery to the urothelial cell layer. No evidence of gene expression was noted in the submucosal and smooth muscle layers. Intravesical gene transfer to bladder epithelium presents several favorable clinical scenarios and it may be used in a number of diseases, such as fibrosis and transitional cell carcinoma. Using direct in vivo transfection genes may easily be transferred to the kidneys and testes percutaneously via a small gauge needle. Repeat bladder treatments through a urethral catheter would also be an ideal approach with this technology. Although the experiments performed in our study revealed transient expression, permanent expression may be possible using appropriate vectors. Nevertheless, several clinical conditions, such as infection and idammation, would best benefit from transient transfection. In these situations application may be repeated. However, to our knowledge it is unknown whether repetitive electrical treatments in an organ may cause tissue damage. It is also unknown how efficient this technique may prove in the diseased organ, in which tissue resistance probably differs from that of normal tissue. Studies addressing these issues are currently being investigated at our laboratory.
ical organs. Successful gene transfer to target cells was confirmed by a series of gene expression assays. Its apparent safety and relative simplicity suggest that direct in vivo electrotransfection may be useful clinically. Dr. Heung J a e Park provided technical support. REFERENCES
1. Friedmann, T.: The maturation of human gene therapy. Acta Paed., 85: 1261, 1996. 2. Kay, M. D., Liu, D. X. and Hoogerbrugge, P. M.: Gene therapy. Proc. Natl. Acad. Sci., 94: 12744,1997. 3. Ferrari, G.: Retroviral vectors for human gene therapy. Minerva Biotecnol., 9 108, 1997. 4. Mulligan, R. C.: The basic science of gene therapy. Science, 260: 926,1993. 5. Lyerly, H. K. and DiMaio, J. M.: Gene delivery systems in surgery. Arch. Surg., 128 1197,1993. 6. Crystal, R. G.: Transfer of genes to humans: early lessons and obstacles to success. Science, 270 404,1995. 7. Miller, A. D.: Human gene therapy comes of age. Nature, 357: 455, 1992. 8. Matthews, K. E., Mills, G. B., Horsfall, W., Hack, N., Skorecki, K and Keating, A,: Bead transfection: rapid and efficient gene transfer into marrow stromal and other adherent mammalian cells. Exp. Hematol., 21: 697,1993. 9. Lasic, D. D. and Papahadjopoulos, D.: Liposomes revisited. Science, 267: 1275,1995. 10. Weaver, J. C.: Electroporation: a general phenomenon for manipulating cells and tissues. J. Cell. Biochem., 51: 426,1993. 11. Chang, D. C.,Gao, P. Q. and Maxwell, B. L.: High efficiency gene transfection by electroporation using a radio-frequency elelctric field. Biochem. Biophys. Acta, 1092 153,1991. 12. Chomezynski, P. and Sacchi, N.: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162 156,1987. 13. Titomirov, A. V.,Sukharev, S. and Kistanova, E.: In vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNA. Biochem. Biophys. Acta, 1088 131, 1991. 14. Nishi, T., Yoshizato, K., Yamashiro, S., Takeshima, H., Sato, K., Hamada, K., Kitamura, I., Yoshimura, T., Saya, H., Kuratsu, J. and Ushio, Y.: High-efficiency in vivo gene transfer using intraarterial plasmid DNA injection following in vivo electroporation. Cancer. Res., 5 6 1050,1996. 15. Aihara, H. and Miyazaki, J.: Gene transfer into muscle by electroporation in vivo. Nat. Biot., 1 6 867,1998. 16. Wagner, J., Madry, H. and Reszka, R.: In vivo gene transfer: focus on the kidney. Nephrol. Dial. Transplant., 1 0 1801, 1995. 17. Wolff, J. A., Malone, R. W., Williams, P, Chong, W., Acsadi, G., Jani, A. and Felgner, P. L.: Direct gene transfer into mouse muscle in vivo. Science, 247: 1465,1990. 18. Davis, H. L., Whalen, R. G. and Demeneix, B. A.: Direct gene transfer into skeletal muscle in vivo: factors affecting efficiency of transfer and stability of expression. Hum. Gene Ther., 4: 151,1993. 19. Hickman, M. A., Malone, R. W., Lehmann-Bruinsma, K., Sih, T. R., Knoell, D., Szoka, F. C., Walzem, R., Carlson, D. M. and Powell, J. S.: Gene expression following direct injection of DNA into liver. Hum. Gene Ther., 5 1477,1994. CONCLUSIONS 20. Moullier, P., Friedlander, G., Calise, D., Ronco, P., Perricaudet, Our study demonstrates that direct in vivo electrotransfecM. and Ferry, N.: Adenoviral-mediated gene transfer to renal tion is a feasible method for gene delivery into intact urologtubular cells in vivo. Kidney Int., 4 5 1220,1994.
DISCUSSION
Dr. Mark Adams. Perhaps I misunderstood your bar graph but it looked to me a s though you had a high level of gene expression in week 1, and then it decreased fairly dramatically at 10 and 14 days. Do you have any long-term data to show that this does not dwindle out altogether and gene therapy was effective? Dr. James J . Yoo. The current limitation of using naked plasmid is transient expression. However, naked plasmids are the safest means of transfection.
Vol. 162, 111S1118,September 1999 Printed in U S A .
THE JOURNAL OF UROLOGY Copyright 0 1999 by AMERICAN UROLOCICAL ASSOCIATION, INC.
DIRECT IN VIVO GENE TRANSFER TO UROLOGICAL ORGANS JAMES J . YOO, SHAY SOKER, LEE F. LIN, KATHRYN MEHEGAN, PAUL D. GUTHRIE AND ANTHONY ATALA* From the Laboratory for Tissue Engineering and Cellular Therapeutics, Department of Urology, Children's Hospital and Harvard Medical School, Boston, Massachusetts
ABSTRACT
Purpose: Patients with urological disorders may benefit from gene based therapy. We investigated the feasibility of delivering exogenous genes into urological tissues in vivo using direct in vivo electrotransfection. Materials and Methods: Gene transfer to rat kidneys, testes and bladders was accomplished via direct local injection of pGL3/luciferase and P-galactosidase reporter gene constructs, followed by a n electrical pulse ranging from 55 to 115 msec. at 100 V. Direct injection of deoxyribonucleic acid without a n electrical pulse served as the control. The transfected and nontransfected organs were retrieved and analyzed by luciferase activity assay, histochemical and immunocytochemical staining for P-galactosidase, and reverse transcription polymerase chain reaction with primers specific for P-galactosidase messenger ribonucleic acid. Results: There was significant luciferase activity 1, 3 and 5 days after direct in vivo electrotransfection in kidneys and testes, and after 3, 5, 7 and 10 days in bladders. Positive P-galactosidase enzyme activity and P-galactosidase immunoreactivity were observed in the transfected renal tubular cells, testicular interstitial and germ cells, and uroepithelial bladder layer. Reverse transcription-polymerase chain reaction products of the transfected organs were noted, indicating the successful transcription of messenger ribonucleic acid. Conclusions: This study demonstrates that direct in vivo electrotransfection is a feasible method of transient gene delivery into intact urological organs. Its apparent safety and relative simplicity suggest that direct in vivo electrotransfection may be useful clinically. KEY WORDS:gene transfer, electroporation, kidney, testis, bladder
Recent advances in genetic engineering and molecular techniques have facilitated the production of various synthesized genes for therapeutic purposes. These technological advances have allowed the initiation of gene targeted therapies in various diseases Methods of in vivo gene based therapy are currently being approached by using viral or nonviral vectors. Gene transfer using viral vectors (retroviral and adenoviral) is achieved by infectivity of the viral particles. Although mammalian viral based vectors have gained wide acceptance, they have been associated with several problems. Retroviral vectors require replicating cells for integration and expression, and they carry only relatively small segments of deoxyribonucleic acid (DNA).4-6 Adenoviral vectors necessitate repeat administration due t o transient expression and they are associated with strong antigenecit^.",^ In contrast, nonviral gene transfer systems using naked plasmid DNA are considered safe and Unlike viral vectors the introduction of plasmid DNA into target cells is facilitated by chemical or physical Electroporation is a physical method of introducing macromolecules into cells in vitro by applying a brief electrical pulse that causes transient molecular membrane transit. This method provides higher transfer efficiency compared with other nonviral transformation methods."-" We developed a method to achieve organ confined gene transfer into urological organs in vivo, which we termed direct in vivo electrotransfection. Based on the principle of electroporation we designed a gene transfer device that would be effective in intact hollow and solid organs. This study was designed to determine the effectiveness of direct in vivo electrotransfection in intact kidneys, testes and bladders.
MATERIALS A N D METHODS
P l a s m i d s . Two vector systems, pGL3 and pCMVp, were
used for the transfection studies. pGL3 contains a reporter gene encoding firefly luciferase expressed by an SV40 promoter enhancer. pCMVp, driven by a cytomegalovirus promoter enhancer, contains the Escherichia coli lacZ gene encoding p-galactosidase. Plasmids were prepared by standard alkaline lysis techniques followed by ethanol precipitation. Phosphate buffered saline was used as the transfection buffer. A n i m a l model a n d gene transfer techniques. A total of 168 male Sprague-Dawley rats were studied, including the kidney, testis and bladder in 56 each. Gene transfer was performed with a device specifically designed for this study. The device was equipped with 2, 560 pF.1250 V. electrolytic capacitors charged in parallel through a limiting resistance to the desired voltage. The capacitors were subsequently disconnected from the power supply and discharged through the organ studied. Gene transfer to rat kidneys, testes and bladders was accomplished via direct local injection with a n 18 gauge silicone catheter. After the introduction of plasmid DNA into each kidney, testis and bladder organ contact was achieved with a negative electrode needle through the sheath. A positive electrode was placed externally. A current of 100 V. was applied to all organs. Mean treatment time plus or minus standard deviation was 89 -+ 6 msec. in kidneys, 105 2 10 in testes and 57 2 3 in bladders (range 55 to 115)depending on the specific tissue resistance. A dose of 100 pg. pGL3 was injected unilaterally into 42 kidneys and 42 testes, and instilled through a catheter into 42 bladders before the electric pulse was applied. Transfected * Requests for reprints: Children's Hospital and Harvard Medical and nontransfected organs (liver, spleen, kidney, testis and bladder) were retrieved on days 0, 1 , 3 , 5 , 7 , 10 and 14 after School, 300 Longwood Ave., Boston, Massachusetts 02115. 1115
1116
DIRECT IN VIVO GENE TRANSFER TO UROLOGICAL ORGANS
transfection for luciferase activity assay analysis (6per time point). Direct injection of DNA without a n electrical pulse served as the control. One organ transfection session was performed per animal. To determine the sites of cellular uptake and expression of the transfected genes kidneys, testes and bladders in another set of animals were transfected with pCMVp, an expression plasmid encoding the gene for E. coli p-galactosidase (lacZ) under the conditions described. Transfection with phosphate buffered saline only (no DNA) served as the control. The 14 testes, 14 kidneys and 14 bladders were retrieved at 1 , 3 and 5 days, respectively, for histochemical, immunocytochemical and reverse transcriptase (RT) polymerase chain reaction (PCR) studies. Luciferase activity assay. The 42 retrieved rat kidneys, 42 testes, 42 bladders and distant organs were frozen in liquid nitrogen. Each organ was homogenized in a tissue grinder and lysed in 1x lysis buffer (25 mM. tris, pH 7.8, with phosphoric acid, 2 mM. 1,2-diaminocyclohexe-N,N,N', tetraacetic acid, 2 mM. dithiothreitol, 10% glycerol and 1% Triton X-100) at room temperature for 15minutes. ARer centrifugation 20 pL. protein lysates were mixed with 100 pL. substrate, consisting of 270 pM. coenzyme A (lithium salt), 470 pM. luciferin and 530 pM. adenosinetriphosphatase. Luciferase activity was measured with a scintillation counter in triplicate in each organ. Statistical evaluation was performed using the Student t test with p 5 0.05 considered significant. Histochemical and imrnuruxytochernical studies. Retrieved organs were thoroughly flushed in phosphate buffered saline and frozen in OCT embedding compound. Cryostat sections (6 jm.were ) fixed in 4% paraformaldehyde for 10 minutes and subsequently stained with X-gal (5-bromo-4-chloro-3-indolyl-~ galahside). Fgalahsidase cleaves this substrate into an indigo compound, such that cells producing the transferred Pgalahsidase gene product are stained blue. Working solution for X-gal consisted of 10 mM. potassium ferricyanide, 10 mM. potassium ferrocyanide, 2 mM. magnesium chloride, 0.02% NF'-40,0.01% sodium deoxycholate and 0.4 mgJml. x-gal in dimethylformamide. Immunocytochemical study was performed with a mouse monoclonal anti-egalactosidase antibody using the avidin-biotin detection system. Subsequently tissue sections were counterstained with hematoxylin. RT-PCR for ribonucleic acid (RNA) analysis. RNA was extracted using a standard TRIzol method with some modifications." Each organ was homogenized in TRIzol reagent and RNA was precipitated with isopropyl alcohol. Subsequently RNA was mixed in a solution consisting of l x PCR buffer, 0.01 units per pl. ribonuclease inhibitor and 0.04 units per pl. deoxyribonuclease I. The reaction mixture was incubated at 37C for 30 minutes. Immediately after incubation 0.25 pg./pl. proteinase K was added to the reaction mixture and incubated again for 30 minutes. After precipi-
tation of RNA complementary DNA synthesis was performed using oligo d(T) primer, as described by the manufacturer. PCR was performed using a n amplification cycle profile consisting of 94C for 1 minute, 62C for 1 minute and 72C for 2 minutes per cycle. After 30 PCR cycles a n additional cycle at 72C for 7 minutes was performed to ensure complete DNA extension. RESULTS
All animals survived without any complications. At retrieval the transfected and distant organs appeared normal grossly and histologically. Luciferase activity in the transfected kidneys was measured (6 per time point). Peak luciferase activity was noted at days 3 and 5 in the electroporated kidneys (fig. 1, A ) . Kidneys injected with DNA without electroporation also showed luciferase activity, although reporter activity was consistently weaker than that in electroporated kidneys (p <0.05). In the transfected testes significant luciferase activity was expressed 1, 3 and 5 days after electrotransfection, and lower levels of activity were detected throughout the remaining course of the study (6 specimens per time point, p <0.05, fig. 1,B). Luciferase activity in the control testes injected with DNA without electrical pulse was not significant. In contrast, the 42 bladders consistently had maximal expression at day 7 (fig. 1, C). Bladders instilled with DNA only as the controls failed to show any activity. Nontransfected organs retrieved from electrically pulsed animals 0, 1,3, 5 7, 10 and 14 days after the procedure consistently failed to demonstrate any luciferase activity greater than the background level. Histochemical and immunocytochemical analyses of P-galactosidase expression in the transfected tissue revealed similar results. In the 8 kidneys positive staining was confined to the renal cortex, while the medulla failed t o stain. Renal tubular cells stained positively but other structures, such as glomeruli, collecting ducts and interstitial cells, did not stain (fig. 2, A and B). In the 8 transfected testes there was positive 6-galactosidase expression in interstitial and germ cells in the lumen of the seminiferous tubules (fig. 2, C and D).In the 8 bladders at day 7 the uroepithelial cell layer stained positive. However, the submucosal and smooth muscle layers failed t o stain (fig. 2, E ) . None of the phosphate buffered saline treated control tissues or distant organs not transfected revealed any P-galactosidase activity. As an independent assay for gene expression, RT-PCR was performed. RT-PCR products of expected size (500 bp) were obtained with RNA from 6 kidneys, 6 testes and 6 bladders transfected with pCMVj3. Control tissues failed to show any p-galactosidase messenger (m) RNA by RT-PCR. These findings indicate that P-galactosidase mRNA was transcribed in the transfected kidneys, testes and bladders (fig. 3).
FIG. 1. Time course of luciferase gene expression after direct in viva electrotransfection (DIVE)with 100 pg. pGL3. A, peak activity in kidneys at days 3 and 5. B , significant activity in testes a t days 1, 3 and 5. C , peak activity in transfected bladders at day 7.
DIRECT IN VIVO GENE TRANSFER TO UROLOGICAL ORGANS
1117
FIG. 2. Histochemical and immunocytochemical analyses of P-galactosidase expression. A, indigo blue staining in renal cortex but not in medulla in transfected kidneys. X-gal staining, reduced from X25. B . 6-galactosidase expression only in renal tubular cells with glomeruli and stromal cells staining negatively. X-gal staining, reduced from X250. C , indigo blue staining in interstitium and germ cells in direct in vivo electrotransfection testis. X-gal staining, reduced from X25. D , immunocytochemical study using monoclonal P-galactosidase antibody shows positively stained germ cells in seminiferous tubule. Reduced from X400. E , immunocytochemical analysis of P-galactosidase expression using p-galactosidase monoclonal antibody in transfected bladders with positive staining over whole urothelial cell layer. Reduced from X100.
1
2
3
4
5
6
7
bP
--600 ---
700
500
---
400
---
FIG. 3. RT-PCR products indicate P-galactosidase mRNA in transfected bladders (2), kidneys ( 4 ) and testes ( 6 ) .Control bladders (3), kidneys (5)and testes (7) failed to show a n y P-galactosidase mRNA. 1 , positive
control p-galactosidase plasmid. DISCUSS I 0 N
Urological disorders of the kidney, testis and bladder may be treated by introducing specific genes in a manner that may correct the malfunctioning cellular components. In this study we explored the possibility of delivering exogenous genes to intact urological organs. For the gene transfer methods we used the principle of electroporation. Electroporation is an established in vitro method of gene transfer in cultured cells with relatively high efficiency.", l1 Recently gene transfer by electroporation has been attempted in several tissues, including the muscle, skin and vessel^.'^-^^ I n this study we designed an electroporation gene transfer device for targeting whole urological organs in vivo. Based on the fact that electrical current flows between electrodes we designed the system to transmit mild electrical pulses to the target organ. Transmission was made possible by discharging electrical current through a central electrode needle toward the other node, thus, encasing the whole organ externally. This technique enabled the current to travel omnidirectionally from the centrally located electrode toward the surface of the target organ. The applied electrical field transmitted by the
direct in vivo electrotransfection device affects the whole organ. This system is an improvement over the in vivo electroporation system used in previous studies, in which the range of electrical current was limited by the area inside the e1e~trodes.l~~~ Histological examination of the electrotransfected organs revealed no evidence of damage subsequent to transfection and there were no apparent systemic or distant organ effects. Various therapeutic modalities involving electrical sources have been applied for clinically managing certain diseases in humans, including cutaneous electrostimulation for pain relief and electroconvulsive therapy in manic depressive disorders. These widely accepted therapeutic applications indicate that controlled exposure of a patient to electrical current is safe. We demonstrated in thisstudy that a current setting of 100 V. is efficient for gene transfer based on the quantitation of reporter gene activity and is within the current settings used in other medical modalities (range 50 to 130 V.). Most importantly current exposure time is limited to msec., providing a virtually painless and rapid procedure. Gene transfer systems using naked plasmid DNA vectors usually result in transient e x p r e ~ s i o n . ~In - ~ our current study using pGL3 there was also a limited duration of gene expression. However, the use of nonintegrating episomal expression vectors that replicate extrachromosomally may allow this procedure to be applied when prolonged expression of the transferred genes is desirable. Safety has been a continued concern in the development of gene therapy protocols. Although a number of clinical trials of various vector systems have been satisfactory, the possibilities of viral infection, mutagenesis and antigenecity may be further decreased by using naked plasmid DNA.We demonstrated that expression of genes may be elicited from nonindependently replicating plasmid DNA, indicating that commonly used recombinant expression vectors may be used in this procedure. Plasmids may also be purified to homogeneity by straightforward techniques, decreasing the risk of immunological reaction to foreign proteins. This latter feature should also allow repeat treatments without the risk of immunological complications. The kidney is an attractive target organ for gene delivery because it may be manipulated by percutaneous methods.16
1118
DIRECT IN VIVO GENE TRANSFER TO UROLOGICAL ORGANS
The direct injection of plasmids resulted in reporter gene expression. This result is consistent with those in previous reports of successful gene transfer in vivo by direct injection of naked DNA into tissues, such as the heart, liver and skeletal muscle. 17-19 Nevertheless, renal gene transfer was enhanced by using direct in vivo electrotransfection. Analysis of Pgalactosidase expression by in situ enzyme assay and immunocytochemical study aRer transfection with a lacZ reporter plasmid indicated that renal tubular cells expressed the transfected gene while other structures, such as glomeruli, collecting ducts and interstitial cells, did not express the gene or expressed it at a lower level. These results resemble those of Moullier et al, who delivered a replication defective adenovirus carrying a lacZ reporter cassette to the kidney by direct infusion into the renal artery.20 P-galactosidase expression was observed in the tubular epithelial cells of the renal cortex with no evidence of gene transfer in glomerular structures, or vascular or interstitial compartments. Although to our knowledge the mechanisms underlying these apparent differences in cell targeting are unknown, these results suggest that tubular epithelial cells, which comprise greater than 90% of the constituent cells of the kidney, may be preferential targets for gene therapy using the direct in vivo electrotransfection method. Our study shows the possibility of introducing foreign genes in testes. P-galactosidase gene expression was detected in various cell types, including interstitial and germ cells. Specific genes regulating tumor cell suppression or killing may be directly delivered t o affected cells using direct in vivo electrotransfection. In the future testicular gene transfer may lead to the production of transgenic animals and may be applied as a treatment strategy for hereditary disease. Gene transfer to bladders using electrotransfection resulted in gene delivery to the urothelial cell layer. No evidence of gene expression was noted in the submucosal and smooth muscle layers. Intravesical gene transfer to bladder epithelium presents several favorable clinical scenarios and it may be used in a number of diseases, such as fibrosis and transitional cell carcinoma. Using direct in vivo transfection genes may easily be transferred to the kidneys and testes percutaneously via a small gauge needle. Repeat bladder treatments through a urethral catheter would also be an ideal approach with this technology. Although the experiments performed in our study revealed transient expression, permanent expression may be possible using appropriate vectors. Nevertheless, several clinical conditions, such as infection and idammation, would best benefit from transient transfection. In these situations application may be repeated. However, to our knowledge it is unknown whether repetitive electrical treatments in an organ may cause tissue damage. It is also unknown how efficient this technique may prove in the diseased organ, in which tissue resistance probably differs from that of normal tissue. Studies addressing these issues are currently being investigated at our laboratory.
ical organs. Successful gene transfer to target cells was confirmed by a series of gene expression assays. Its apparent safety and relative simplicity suggest that direct in vivo electrotransfection may be useful clinically. Dr. Heung J a e Park provided technical support. REFERENCES
1. Friedmann, T.: The maturation of human gene therapy. Acta Paed., 85: 1261, 1996. 2. Kay, M. D., Liu, D. X. and Hoogerbrugge, P. M.: Gene therapy. Proc. Natl. Acad. Sci., 94: 12744,1997. 3. Ferrari, G.: Retroviral vectors for human gene therapy. Minerva Biotecnol., 9 108, 1997. 4. Mulligan, R. C.: The basic science of gene therapy. Science, 260: 926,1993. 5. Lyerly, H. K. and DiMaio, J. M.: Gene delivery systems in surgery. Arch. Surg., 128 1197,1993. 6. Crystal, R. G.: Transfer of genes to humans: early lessons and obstacles to success. Science, 270 404,1995. 7. Miller, A. D.: Human gene therapy comes of age. Nature, 357: 455, 1992. 8. Matthews, K. E., Mills, G. B., Horsfall, W., Hack, N., Skorecki, K and Keating, A,: Bead transfection: rapid and efficient gene transfer into marrow stromal and other adherent mammalian cells. Exp. Hematol., 21: 697,1993. 9. Lasic, D. D. and Papahadjopoulos, D.: Liposomes revisited. Science, 267: 1275,1995. 10. Weaver, J. C.: Electroporation: a general phenomenon for manipulating cells and tissues. J. Cell. Biochem., 51: 426,1993. 11. Chang, D. C.,Gao, P. Q. and Maxwell, B. L.: High efficiency gene transfection by electroporation using a radio-frequency elelctric field. Biochem. Biophys. Acta, 1092 153,1991. 12. Chomezynski, P. and Sacchi, N.: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162 156,1987. 13. Titomirov, A. V.,Sukharev, S. and Kistanova, E.: In vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNA. Biochem. Biophys. Acta, 1088 131, 1991. 14. Nishi, T., Yoshizato, K., Yamashiro, S., Takeshima, H., Sato, K., Hamada, K., Kitamura, I., Yoshimura, T., Saya, H., Kuratsu, J. and Ushio, Y.: High-efficiency in vivo gene transfer using intraarterial plasmid DNA injection following in vivo electroporation. Cancer. Res., 5 6 1050,1996. 15. Aihara, H. and Miyazaki, J.: Gene transfer into muscle by electroporation in vivo. Nat. Biot., 1 6 867,1998. 16. Wagner, J., Madry, H. and Reszka, R.: In vivo gene transfer: focus on the kidney. Nephrol. Dial. Transplant., 1 0 1801, 1995. 17. Wolff, J. A., Malone, R. W., Williams, P, Chong, W., Acsadi, G., Jani, A. and Felgner, P. L.: Direct gene transfer into mouse muscle in vivo. Science, 247: 1465,1990. 18. Davis, H. L., Whalen, R. G. and Demeneix, B. A.: Direct gene transfer into skeletal muscle in vivo: factors affecting efficiency of transfer and stability of expression. Hum. Gene Ther., 4: 151,1993. 19. Hickman, M. A., Malone, R. W., Lehmann-Bruinsma, K., Sih, T. R., Knoell, D., Szoka, F. C., Walzem, R., Carlson, D. M. and Powell, J. S.: Gene expression following direct injection of DNA into liver. Hum. Gene Ther., 5 1477,1994. CONCLUSIONS 20. Moullier, P., Friedlander, G., Calise, D., Ronco, P., Perricaudet, Our study demonstrates that direct in vivo electrotransfecM. and Ferry, N.: Adenoviral-mediated gene transfer to renal tion is a feasible method for gene delivery into intact urologtubular cells in vivo. Kidney Int., 4 5 1220,1994.
DISCUSSION
Dr. Mark Adams. Perhaps I misunderstood your bar graph but it looked to me a s though you had a high level of gene expression in week 1, and then it decreased fairly dramatically at 10 and 14 days. Do you have any long-term data to show that this does not dwindle out altogether and gene therapy was effective? Dr. James J . Yoo. The current limitation of using naked plasmid is transient expression. However, naked plasmids are the safest means of transfection.