Jan 26, 1993 - 21, No. 15 3567-3573. Electroporation enhances c-myc antisense ... resuspended in sterile water. Aliquots were resolved on a .... capped FITC-c-myc-as ODN was added to K562 cells and cell associated ..... 20, 5691 -5698. 10. ... Evan,G.I., Lewis,G.K., Ramsay,G. and Bishop,J.M. (1985) Mol. Cell. Biol.
Nucleic Acids Research, 1993, Vol. 21, No. 15 3567-3573
Electroporation enhances c-myc antisense oligodeoxynucleotide efficacy Raymond Bergan, Yvette Connell, Brigid Fahmy and Leonard Neckers Clinical Pharmacology Branch, NCI, NIH, Bethesda, MD 20892, USA Received January 26, 1993; Revised and Accepted May 14, 1993
ABSTRACT Obtaining high transfection efficiencies and achieving appropriate intracellular concentrations and localization are two of the most important barriers to the implementation of gene targeted therapy. The efficiency of endogenous uptake of oligodeoxynucleotides (ODNs) varies from cell type to cell type and may be a limiting factor of antisense efficacy. The use of electroporation to obtain high intracellular concentrations of a synthetic ODN in essentially 100% of viable cells is described. It is also shown that the transfected ODNs initially localize to the nucleus and remain there for at least 48 hours. The cellular trafficking of electroporated ODNs is shown to be an energy dependent process. Targeting of the c-myc proto-oncogene of U937 cells by electroporation of phosphorothioate-modified ODNs results in rapid and specific suppression of this gene at ODN concentrations much lower than would otherwise be required. This technique appears to be applicable to a variety of cell types and may represent a powerful new investigative tool as well as a promising approach to the ex vivo treatment of hematologic disorders.
INTRODUCTION Strategies aimed at the genetic manipulation of bone marrow in an ex vivo setting are avidly being sought (1, 2, 3, 4). Some of these approaches involve the use of synthetic oligodeoxynucleotides (ODNs) to target specific genes in an antisense fashion. Among the most formidable barriers to the in vivo or ex vivo use of ODNs are the attainment of uniform cell permeation and appropriate intracellular targeting. Although there are a number of techniques available to enhance cellular uptake of synthetic ODNs and of DNA- and RNA-based vectors, inefficiency and harsh conditions remain a problem (5, 6, 7). The greatest success with large pieces of DNA/RNA has been obtained with retroviral vectors where transfection efficiencies of 60 to 100% are common through the use of antibiotic resistance preselection (1, 2). However, without such preselection typical transfection efficiencies are on the order of 5 to 25 % in hematopoietic cells. There has been some success in augmenting ODN uptake through the use of liposomal encapsulation (8, 9). Electroporation of plasmids into bone marrow derived hematopoietic progenitor cells has been described (10, 11). Electroporation allows delivery of the transfected species directly
into the cytoplasm (12). This is an important consideration when synthetic ODNs are being employed since after endogenous uptake by the cell ODNs are primarily confined to endocytotic vesicles (13, 14). This paper describes the use of electroporation to markedly enhance cellular uptake of synthetic ODNs in an efficient, effective, and reproducible manner. The cellular pharmacokinetics of these ODNs after electroporation are characterized and the ability of ODNs so introduced into cells to specifically target genetic sites in an antisense manner is reported.
MATERIALS AND METHODS Cell cultures K562, U937, PC-12, U-87MG, Daoy, and murine monoclonal c-myc 1-9E10.2 antibody producing hybridoma cells were purchased from the American Type Culture Collection (Rockville, MD). KBM-5 cells were derived from a patient with chronic myelogenous leukemia in blast crisis and were a generous gift from S.O'Brien (MD Anderson) (15). PC-3M and TC-32 cells were generous gifts from J.Trepel and L.Whitesell, respectively, (National Institutes of Health). Cells were cultured in RPMI 1640 with 10% heat inactivated fetal bovine serum (GIBCO BRL Laboratories, Grand Island, NY) and were maintained at 37°C, 6% carbon dioxide with biweekly media changes.
Oligodeoxynucleotide synthesis Oligodeoxynucleotides (ODNs) were synthesized by cyanoethyl phosphoramidite chemistry on an Applied Biosystems model 380B DNA synthesizer. Sulfurizing reagent (Glen Research, Sterling, VA) was used according to manufacturer's instructions. ODNs were ethanol precipitated, washed in 70% ethanol, and resuspended in sterile water. Aliquots were resolved on a denaturing polyacrylamide gel to check for homogeneity. Antisense and scrambled ODNs targeting c-myc mRNA were synthesized fully phosphorothioated. Antisense 15-mer c-myc (c-myc-as) ODNs were of the sequence 5 '-AACGTTGAGGGGCAT-3' which corresponds to the region of c-myc mRNA at the AUG start codon and thus spans coding bases + 1 to + 15. The c-myc scrambled (c-myc-scr) ODNs were of the sequence 5'-CTGAAGTGGCATGAG-3'. These reagents are used to test the biological efficacy of electroporated ODNs. Fluorescein-conjugated c-myc-as ODN contains fluorescein within the sequence as follows: 5'-AACGTTG-
3568 Nucleic Acids Research, 1993, Vol. 21, No. 15 AGGG-X-GCAT-3'. This ODN was synthesized as allphosphodiester, all-phosphorothioate, and phosphorothioatecapped species. These reagents are used in the experiment described in Figure 1. ODN- 1 is a fluoresceinated, phosphorothioate-capped ODN with the sequence 5'-XGTCCACCATGGCGCGGCCGGG-3'. This reagent is used in the
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remaining experiments. Fluorescein-conjugated ODNs were synthesized using cyanoethyl phosphoramidite chemistry with fluorescein phosphoramidite (Glen Research) following manufacturer's instructions. Phosphorothioate-capped ODNs contain two interbase phosphorothioate linkages at each end of the ODN. Electroporation Electroporation was performed with a GIBCO BRL Cell-Porator Electroporation System 1. For U937 cells capacitance and voltage were set at 1180 microferads (uLF) and 280 volts (V) respectively; for all other cell types the Cell-Porator was set at 800 ltF and 300 V. Cells were electroporated in 0.4 cm gap electroporation chambers (GIBCO BRL, Gaithersberg, MD). U937 cells were electroporated in lml of Dulbecco's modified essential medium without fetal bovine serum at a maximum concentration of 4 x 106 cells per ml; after electroporation fetal bovine serum was added to a final concentration of 10%. All of the other cell types were electroporated in 1 ml of RPMI 1640 with 10% fetal bovine serum. The maximum concentration of K562 cells during electroporation was 5 x 106 cells per ml and the maximum concentration of all other cell types was 1 x 106 cells per ml. After electroporation cells were maintained at 37°C, unless otherwise specified.
Cell lysis and Western blotting One to two million cells were lysed in cell lysis buffer (50 mM Tris, pH 7.5; 1% NP-40; 2 mM EDTA; 100 mM NaCl) with 20 itg/ml leupeptin, 1 Ag/ml aprotinin, and 1mM phenylmethylsulfonylfluoride as protease inhibitors. After clarification at 14,000 xg, 4°C, for 20 minutes, protein extracts from an equal number of cells were separated on an 8% sodium dodecyl sulfate polyacrylamide gel. Western transfer was performed as described previously with modifications (16). Briefly, transfer was performed in a semidry transfer apparatus (Pharmacia) in glycine transfer buffer (40 mM glycine, 50 mM Tris, pH 8.8, 0.1% sodium dodecyl sulfate, 20% methanol (v/v)) for two hours at 0.8 milliampere/cm2 to 0.45 ltm pure nitrocellulose (Schleicher and Schuell Inc.). Western blotting was carried out with the ECL Western blotting kit (Amersham) according to manufacturer's instructions. Briefly, membranes were blocked in 5 % non-fat dry milk in TTBS (20 mM Tris HCI, pH 7.6; 0.05% Tween 20; 0.9% sodium chloride) for 1 hour, incubated with undiluted cmyc mouse monoclonal antibody-containing conditioned medium for 1 hour, with sheep anti-mouse horseradish peroxidase-linked antibody diluted 1:1000 for 1 hour, and then lastly incubated in a luminol based detection solution for 1 minute. Membranes were then exposed for various times to Kodak X-OMAT AR film. Flow cytometric analysis Flow cytometric (FACS) analysis of cells containing fluorescent ODNs was performed as described previously (17).
RESULTS Electroporation enhances cellular ODN uptake The marked enhancement of cell associated fluorescence that can be achieved with electroporation is illustrated in Figure 1. Twenty ,uM of phosphorothioate, phosphodiester, or phosphorothioatecapped FITC-c-myc-as ODN was added to K562 cells and cell associated fluorescence was followed by FACS analysis in electroporated and control cells over time. Due to the transient
Nucleic Acids Research, 1993, Vol. 21, No. 15 3569 when compared to unelectroporated cells. Furthermore, there is a notable difference in subcellular localization. After electroporation most of the ODN is located in the nucleus with a smaller amount located within the endocytic vesicles and the remainder being located in the cytoplasm. At twenty-four hours the majority of intracellular ODN remains predominately nuclear though now there is also a larger intravesicular component. In contrast, in unelectroporated cells at one hour the majority is located in endocytic vesicles with only a small amount located in the nucleus. Even after twenty-four hours exposure of unelectroporated cells to ODN, the bulk of the ODN is still located in vesicles with only a small nuclear component. After three days the state of intracellular ODN in electroporated and unelectroporated cells is equivelent (Figure 2F). Essentially all of the ODN is contained within intracytoplasmic vesicles, a small amount is in the cytoplasm, and virtually none is in the nucleus.
fragility of cells after electroporation, manipulations of the cells within one hour after electroporation was limited. One hour after electroporation it can be seen that there is a marked elevation of cell associated fluorescence with all three types of backbone modification. The electroporation of ODNs with various lengths, all less than 22 bases, has been performed in our laboratory with similar results (data not shown). There is an initial rapid decrease with time of the cell-associated fluorescence seen in the cells electroporated with both phosphodiester and phosphorothioatecapped ODNs. Cell-associated fluorescence approaches unelectroporated values within 8 hours in the phosphodiester treated cells (Figure IA). However, in the case of phosphorothioate-capped treated cells, there remains a difference between electroporated and unelectroporated cells even after 24 hours (Figure 1B). In the case of the all-phosphorothioate treated cells an ongoing increase in cell-associated fluorescence occurs following electroporation (Figure 1C). To demonstrate that the cell-associated fluorescence observed represents intracellular ODN, cellular fluorescence was assessed after washing with 0.2 M glycine, pH 4.0, a maneuver which will remove surface bound ODN. There was no change of fluorescence in electroporated or unelectroporated K562 cells when compared to those that were not acid washed (data not shown). Fluorescence microscopic analysis of cytospin preparations confirms that the ODN is localized intracellularly (Figure 2). Comparison of the one hour photomicrographs reveals that in electroporated cells there is a marked increase of ODN
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Cellular efflux of electroporated phosphorothioate-capped ODN is energy dependent The rapid decrease in cell-associated fluorescence following electroporation with phosphodiester ODN (see Figure 1) is most likely a reflection of the susceptibility of phosphodiesters to rapid intracellular degradation (data not shown and 18). However, the discrepancy observed between the intracellular trafficking of fully phosphorothioated and phosphorothioate-capped ODNs cannot be attributed to ODN degradation since minimal intracellular
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Figure 2. Photomicrographs of K562 cells at various times after exposure to 20 AM ODN-1: without (top row) and with (bottom row) electroporation. A) 15 min; B) and D) 1 hr; C) and E) 24 hrs; F) 72 hrs.
3570 Nucleic Acids Research, 1993, Vol. 21, No. 15 degradation of phosphorothioate-capped ODNs is seen even after 48 hours (data not shown and 18). In order to study these divergent trafficking patterns, the energy dependent nature of ODN efflux was evaluated. If cellular efflux of electroporated phosphorothioate-capped ODNs is dependent upon a concentration gradient across the plasma membrane, then
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Flgure 3. Change in cellular fluorescence in K562 cells after exposure to ODN-1 under various conditions. A) Effect of extracellular ODN concentration upon cellular efflux of electroporated ODN. Two hours after cells were electroporated in the presence of 10 zM ODN-1 they were maintained either in the presence of ODN-1 or placed into fresh media without ODN. Remaining fluorescence at five hours, as a percentage of fluorescence at two hours, was then determined. iat B) Energy dependence of cellular uptake. Fluorescence was detemined ly after adding 10 zM ODN-1 (baseline) and then at 1 and 5, hours while cells were maintained either at 3°C or at 4°C with 0.1% sodium azide. C) Energy dependence of cellular efflux of electroporated ODN. Fluorescence was determined 1.5 hours after electroporation in the presence of 10 /AM ODN-1 (baseline) and then again at 4 hours while being maintained as in Figure 3B. Reported values are mean +/ - SE of one experiment run in triplicate and are in agreement with results obtained in a previous experiment, also run in triplicate.
changes in the concentration gradient should affect efflux. The lack of dependence of cellular efflux upon a concentration gradient was demonstrated by the following experiment: a high intracellular concentration of ODN was attained by electroporating K562 cells in the presence of 10 jiM ODN-1. Two hours later baseline fluorescence was determined by FACS analysis. The cells were then either maintained in 10 ItM ODN-1 or were washed and maintained in media without ODN-1. Three hours after this, cellular fluorescence was again determined. Figure 3A shows that there is essentially no difference in the decrease of cellular fluorescence between the cells maintained in 10 ltM ODN-1 and those maintained in media without ODN. It is unlikely that the observed decrease in cellular fluorescence seen over this three hour time interval is due to ODN degradation. ODNs are believed to enter cells through an energy-dependent endocytotic process after which they are primarily localized to endocytic vesicles (8, 19, 20). The majority of ODN electroporated into cells is located in the nucleus and cytoplasm and not in endocytic vesicles. If cellular efflux of electroporated ODNs located in the nucleus and cytoplasm is energy dependent, then efflux should be inhibited by metabolic poisons. To confirm that phosphorothioate-capped ODN uptake is an energy-dependent process in K562 cells, 10 ItM ODN-1 was added exogenously and the cells were then maintained either at 37°C or at 4°C in the presence of 0.1% sodium azide. Changes in cellular fluorescence with time were then determined by FACS analysis. Figure 3B shows that there was no uptake of ODN in cells that were metabolically inhibited even after 5 hours of exposure to ODN-1, whereas uptake is seen in the control cells at 1 hour. These results confirm those of Loke at al. (13). Next, we demonstrated that efflux of electroporated phosphorothioate-capped ODN is also energy dependent by first electroporating ODN-1 into K562 cells and then determining the baseline fluorescence 1.5 hours after electroporation. Cells were then maintained either at 37°C or at 4°C in the presence of sodium azide. Figure 3C shows no decrease in cellular fluorescence after 2.5 hours in cells that were metabolically inhibited.
Determination of the efficiency of ODN electroporation To determine the optimum conditions for ODN electroporation, K562 cells were electroporated in the presence of 20 ,uM ODN-1 at increasing voltages; the resultant transfection curve is shown in Figure 4. It can be seen that the percent of transfected cells plateaus at 90% above 300 volts. By assessing trypan blue exclusion and by two color FACS analysis with ODN-1 and propidium iodide we confirmed that the nontransfected cells are nonviable (data not shown). Thus essentially 100% of viable cells can be transfected. Also notable is that even after the 100% transfection level is reached, further increases in voltage lead to continued increases in the amount of ODN per cell. This is consistent with a larger pore size being induced in the plasma membrane with increasing field strengths during electroporation (12). To demonstrate that electroporation of synthetic ODNs can be achieved in a variety of tissue types, several cell lines were subjected to electroporation in the presence of 10 to 40 IZM ODN. As can be seen in Table 1 a wide variety of tissue types are amenable to such a manipulation. When compared to the uptake of exogenously added ODNs, 3.4 to 27 fold increases in intracellular ODN concentration can be achieved.
Nucleic Acids Research, 1993, Vol. 21, No. 15 3571 Electroporation enhances c-myc antisense efficacy The site of action of synthetic ODNs in causing an antisense effect is not yet clear, nor has their intracellular fate been clearly elucidated (19). There is evidence that such effects may take place in the nucleus via inhibition of processing and/or nuclear export. Our data indicate that with electroporation ODN uptake can be markedly enhanced. Not only is there disproportionate nuclear targeting, at least initially, but there is also a relative paucity of ODN located witiin endocytic vesicles. If ODN degradation takes place witiin such vesicles, as is currently believed to be the case, 1 00
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then electroporation should not only result in a lower concentration of extracellular ODN being required to produce a particular antisense effect, but also in a hastening of the onset of the antisense effect. This hypothesis was tested on c-myc protein synthesis in U937 cells. It has previously been shown that an antisense effect can be achieved using ODNs directed to the translation initiation region of the c-myc message, but this effect takes many hours to days to become apparent (7, 21, 22, 23, 24, 17). U937 cells were exposed to increasing concentrations of antisense and scrambled phosphorothioate ODN and either incubated directly or electroporated. Figure 5 shows the results of western analysis performed 1.5 and 24 hours after exposure to ODN. As the half life of c-myc is between 20 and 30 minutes in most cells, evaluation at 1.5 hours represents three half lives. At this time specific reductions in c-myc protein are evident at the 10 and 50 ,uM concentrations as compared to control and scrambled sequences. At 24 hours there is almost total absence of c-myc in the electroporated cells at the 50 AM level and furither decreases at the 10 ,uM level as compared to the 1.5 hour 10 ltM sample, while there is no decrease in the unelectroporated cells. It has been previously shown by our laboratory that after 72 hours of exposure to c-myc-antisense ODN, an antisense effect is only seen at ODN concentrations above 50 ,aM in U937 cells (17).
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Table 1. Increase in cellular fluorescence following electroporation in various cell types
Figure 4. Transfection curve. K562 cells were electroporated at 800 uF at various voltages in the presence of 20 IsM ODN-1 one hour prior to FACS analysis. Cell associated fluorescence, closed circles; percent positive cells, open circles. Reported values are mean +/- SE of one experiment run in duplicate and are in agreement with results obtained in a previous experiment, also run in duplicate. Un C 3
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Figure 6. Percent of viable (trypan blue excluding) U937 cells 24 hours after electroporation in the presence of c-myc-as or c-myc-scr ODN as compared to U937 cells without ODN added. Data are standardized to viability of untreated cells. Data depicted are the mean +/- SE of two independent experiments.
3572 Nucleic Acids Research, 1993, Vol. 21, No. 15 In contrast, with electroporation of c-myc-as ODN into U937 cells there is an almost instantaneous onset of action and a reduction of at least 5 fold in the threshold ODN concentration necessary for an antisense effect. The antiproliferative action of c-myc-as ODNs upon various cell types has ben well documented. Such effects are reported to occur 2 to 3 days after exposure to exogenously added ODN (7, 21, 23, 24, 17). To assess whether such antiproliferative effects are seen after electroporation, cell viability was determined by trypan blue exclusion 24 hours after electroporation of c-myc as and c-myc scr ODNs. As can be seen in Figure 6, there is a dose dependent decrease in U937 viable cell number 24 hours after electroporation with c-myc-as but not with c-myc-scr ODNs.
DISCUSSION Endogenous uptake of ODNs is a time and energy dependent process (13, 18, 19, 25, 26, 27). Thus to achieve a particular antisense effect long periods of in vitro exposure to relatively high concentrations of ODNs may be required. Herein is described a means of bypassing the cell membrane and targeting ODNs to the nucleus while sparing their initial transit through the cellular vacuolar system. With electroporation, increases in intracellular ODN concentration from 3.4 to 27 fold have been achieved, depending on the cell type studied. Reproducible transfection efficiencies approaching 100% have been demonstrated. Such efficiency is highly desirable from the pharmacokinetic standpoint when clinical applications are being pursued. Such high efficiencies have previously only been described with antibiotic preselection using retroviral vectors. In most systems ODNs are thought to enter cells primarily by endocytosis (13, 19, 28) and are initially restricted to the cellular vacuolar system. From here a small percentage escapes and of this most is believed to be transported to the nucleus (19). The fate of that retained within the vesicles is unknown, but current evidence suggests that continued degradation of the ODN occurs. Wu-Pong, however, has suggested the presence of an anion channel-mediated uptake process (27). Based upon the immunofluorescence patterns obtained after electroporation of phosphorothioate-capped ODNs it appears as though not only is the majority of ODN not localized to the vacuolar system, but that it is directly transported and maintained in the nucleus for at least 48 hours. During this time period there is ongoing endocytosis of exogenous ODNs. This pattern of rapid nuclear localization of ODNs introduced into the cell by bypassing the plasma membrane has been reported before with cellular microinjection experiments (29, 30). In such experiments ODN was found to appear in the nucleus seconds to minutes after
injection.
Through comparative time course evaluations of cellular uptake of phosphodiester, phosphorothioate, and phosphorothioatecapped ODNs, the pharmacologic advantages of an allphosphorothioate ODN have been demonstrated. Specifically, phosphorothioate ODNs are selectively maintained at high intracellular concentrations and are resistant to nuclease attack. The advantage of such agents being intracellularly localized with a biologically significant half life while the extracellular milieu can be cleared of ODN are self evident when an ex vivo application is being considered. The reason for the differential handling of ODNs with various backbone modifications is notyet clear. Current evidence suggests that susceptability to endo- and exonuclease attack may play an
important role in such processes. The susceptibility of allphosphodiester ODNs to exonuclease attack has been well documented and no doubt contributes to the rapid removal of this class of ODN from the cell (personal observations and 19). The moderately rapid disappearance of phosphorothioate-capped ODNs from the cell is harder to explain since these ODNs are stable for at least two days intracellularly (personal observations and 18). However, single stranded endonuclease activity has been identified in mammalian cells and may be playing some role here (31). The nuclear efflux of this species of ODN seems to be energy dependent, implying the involvement of a specific transport system or systems in this process. Why allphosphorothioate ODNs do not appear to utilize this efflux mechanism is currently under investigation. The ability of electroporation to shorten the time of onset of an antisense effect and to lower the threshold concentration of ODN necessary to achieve such an effect has been demonstrated. In U937 cells c-myc protein suppression is seen within 1.5 hours after electroporation. Since c-myc has a half life of approximately 30 minutes, this implies an almost instantaneous onset of action. This effect is also shown to occur at concentrations lower than would be necessary with conventional treatment. Finally, we have shown that the previously demonstrated effects of c-myc-as ODN treatment on cellular viability can also be seen after electroporation. Microinjection has been used to bypass the cell's normal uptake process, but this is technically limited to small numbers of cells (29, 30). Encapsulating ODNs in liposomes has also shown promise in aiding their cellular uptake (8,9). With electroporation, millions of cells can be treated at the same time and antisense effects can be monitored within 90 minutes of treatment. This technique will be useful both as an investighative tool and potentially as an aid to the aclinical administration of ODNs ex vivo.
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