(CANCER RESEARCH 55, 2024-2028, May 15, 1995)

Advances in Brief

Neoplastic Reversion Accomplished by High Efficiency Adenoviral-mediatedDelivery of an Anti-ras Ribozyme

M. Feng, G. Cabrera, Jessy Deshane, Kevin J. Scanlon, and David T. (..'urici'

Gene Therapv Program, Universit\ of Alabama al Birmingham. Birmingham, Alabama 352V4 IM. F., G. C.. J. D.. D. C.¡,and Cit\ tif Hupe National Medical Center, Diiarle,California 9/0/0 ¡K.S.¡

Abstract

Strategies have been developed to abrogate the aberrant expression ofdominant oncogenes as a means to accomplish targeted tumor eradication.We have demonstrated previously the utility of this approach using ahammerhead ribozyme designed to cleave the mutant sequence in codon12 of the activated H-ras oncogene transcript. To develop this strategy into

a practical means to approach malignant disease, methods must be developed to accomplish high efficiency delivery of the ribozyme to targetneoplastic cells. To accomplish this, a recombinant adenovirus was designed that encoded a gene cassette for the H-ras ribozyme. By using this

virus, it was possible to accomplish high efficiency reversion of the neoplastic phenotype in mutant H-ras expressing tumor cells without the needfor any selection steps. The demonstration of the utility of adenoviral-

mediated delivery of anticancer ribozymes will allow the practical development of gene therapy strategies on this basis.

Introduction

Gene therapy strategies have been developed based upon specificcorrection of the molecular lesions etiologic of malignant transformationand progression (1-6). In this regard, distinct strategies of mutation

compensation are directed toward ablation of overexpressed genes orgene products, as in the case of targeting dominant oncogenes, or augmenting deficient wild-type function, in the instance of mutated tumor

suppressor genes. In the former context, a number of methods have beendeveloped to allow specific abrogation of the overexpressed dominant-

oncogene function. These methods include intracellular antibodies, dominant-negative mutations, and antisense methodologies (7-15). One ad

vantage of the method of antisense gene inhibition is that this strategyoffers the potential to achieve oncogene ablation at a proximal level ofgene expression (7-13). Furthermore, antisense oligonucleotides possess

ing a catalytic activity may be exploited to augment the stoichiometry ofgene ablation via sequence-specific cleavage of the target transcript(16-20). To this end, we have explored the utility of a hammerhead

ribozyme designed to cleave the GUC sequence in codon 12 of theactivated H-ras oncogene mRNA transcript (21-24).2 Previous studies

have demonstrated that clonal derivatives of H-ras-transformed tumorcells may be established whereby stable expression of the H-ras ri

bozyme can suppress the neoplastic phenotype.Whereas these studies have demonstrated the potential antitumor

efficacy of ribozyme-mediated oncogene ablation to be of utility for

gene therapy interventions, methods must be developed to mediatehigh efficiency delivery of the ribozymes to tumor targets. Strategiesto exploit ribozyme methods to date have used approaches involving

Received 2/9/95; accepted 4/4/95.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicale this fact.

' To whom requests for reprints should be addressed, at Gene Therapy Program,

University of Alabama at Birmingham, 1824 6th Avenue South, Room WTI 620,Birmingham, AL 35294-3300.

- M. Kashani-Sabet and K. J. Scanlon. Application of ribozymes to cancer gene

therapy, submitted for publication.

selection of stably transfected clones similar to studies from ourlaboratory. In this regard, several groups have proposed the use ofanti-HIV ribozymes to achieve genetic immunization of bone marrowprogenitor cells (25-30). The utility of this approach is based upon the

ability to modify the target cells ex vivo such that stable clones maybe derived expressing the anti-HIV ribozymes. As this approach is of

limited utility for the application of disseminated neoplastic disease,alternative approaches have also been explored. In this regard, liposome vectors have been studied to achieve high efficiency delivery ofribozymes to tumor targets (20). These studies demonstrated thatDOTAP/ribozyme3 complexes were capable of mediating a level of

phenotypic reversion in target cells. Observed levels of gene transfer,however, were too low to allow clinical translation of this vectorapproach presently. Recently, the utility of adenoviral vectors inmediating high efficiency gene transfer has been explored in a varietyof contexts (31-33). For anticancer gene therapy, high efficiencydelivery of wild-type tumor suppressor genes has been shown by

several groups to allow phenotypic reversion of tumor targets in vitroand in vivo (34-36). Based upon these studies, we explored the utility

of adenoviral vectors to achieve efficient delivery of ribozyme. Ourresults demonstrate that this vector system is capable of achievinghigh efficiency transient expression of the anti-H-ras ribozyme in

tumor targets. The magnitude of ribozyme transfer was sufficient toachieve neoplastic reversion in a population of H-ras-transformed

bladder carcinoma cells. This vector thus possesses the attributespotentially allowing the application of ribozyme-mediated approaches

in strategies to accomplish gene therapy of cancer.

Materials and Methods

Cells. The human bladder carcinoma cell line EJ was obtained from American Type Culture Collection (Rockville, MD). The low passage EIAtranscomplementing cell line 293 was obtained from F. Graham (McMasterUniversity, Hamilton, Ontario, Canada). Both cell lines were grown in DMEM(GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum(PAA, Long Beach, CA) and penicillin (100 IU/ml)-streptomycin (25 fig/ml;

GIBCO; complete medium). For studies of cell growth kinetics, cells wereseeded at a density of 5 X IO4 cells in 35-mm dishes. At various times after

treatment, cells were harvested, and viable cells were counted by trypan blueexclusion. In addition, cell viability was determined using the 3-(4,5-dimeth-ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Promega, Madison,

WI). For this MIT assay, cells were plated at a density of 5000 cells/well in96-well clusters. At various times after treatment, cells were analyzed for

released formazan by measuring the absorbance at 490 nm. A standard curvewas done in parallel for each analysis by linear dilutions of nontreated cells.

Recombinant Adenoviral Vector Construction. The H-ras ribozyme wasprepared as described previously (24). A 51-bp fragment with flanking Sailand ///ndlll restriction sites was excised from the plasmid DNA pHßAPr-1-

neo and cloned into the corresponding site within the polylinker of theadenoviral shuttle vector pACCMVpLpARS (+) (graciously provided by

The abbreviations used are: DOTAP, /V-[l-(2,3-dioleoyloxy)propyI]-AyVJV-tri-methyl-aminoniummethyl sulfate: PFU, plaque-forming units; PGK, phosphoglycerate

kinase.

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NEOPLASTIC REVERSION BY DELIVERY OF ANTI-ras RIBOZYME

Dr. R. Gerard, University of Texas, Southwestern, Houston, TX). This plasmidprovides promotion/initiation signals derived from the human cytomegalovirusearly promoter/enhancer and polyadenylation signals from SV40 (Fig. 1). Theresulting shuttle plasmid pACH-ras/RZ was then used to derive an El-deleted,replication-defective recombinant adenovirus using standard methodologies

(37). Briefly, the shuttle plasmid plus the adenoviral packaging plasmid pJM 17(graciously provided by Dr. F. Graham, McMaster University) were cotrans-

fected into the EIA transcomplementing cell line 293 using the commercialcationic liposome vector DOTAP (Boehringer-Mannheim, Mannheim, Germa

ny). Transfected cells were maintained until the onset of cellular cytopathiceffects. The newly generated recombinant adenovirus was plaque purifiedthree times by a standard method. Evaluation of each plaque was then accomplished by direct plaque analysis using PCR. For this analysis, primers weredesigned with specificity for the cytomegalovirus promoter and the H-rasribozyme insert (PI, 5'-GCGTGTACGGTGGGAGGTCT; P2, 5'-GTT-

TCGTCCTCACGGACTCAT); in addition, primers were designed with specificity for the wild-type adenoviral EIA region, predicted to be deleted in theconstruction of the EIA(-) recombinant adenoviral vector (P3, 5'-ATTAC-CGAAGAAATGGCCGC; P4, 5'-CCCATTTAACACGCCATGCA). PCR reactions were carried out using 30 thermal cycles of 94°Cfor 1 min, 56°Cfor1 min, and 72°Cfor 2 min. PCR products were then analyzed by electrophore-

sis on agarose gel. After initial validation using the aforementioned PCRanalysis, the recombinant adenovirus encoding the H-ras ribozyme, rAdGTo,

was expanded on 293 cells and purified by CsCI gradient centrifugation usingstandard methods (38). Purified genomic DNA derived from CsCI-fractionated

recombinant adenovirus was then subjected to restriction endonuclease digestion and electrophoresis on agarose gel to confirm its structural configuration.As a control in each experiment, two kinds of El-deleted recombinant adeno

virus rAdGTl and AdCMVLacZ were used. rAdGTl contains a cytoplasmicform of an anti-erbB-2 single-chain antibody (sFv) that has been described

EcoRI Kpnl BamHI Xbal Sail Hmdlll

CMV promotAd5(0-1.3m.u,

V40 Splice/PolyA

Ad5(9.3-17m.u.)

- Sali VOMÃŒHmdlll

H-ras ribozyme ( 51 bp)

CMV promot

AdS

H-ras ribozyme

SV40 Splice/PolyA

Ad5(3.7m.u.)

pACH-ras/RZ

9.0kb XAd5(0-100m.u.)

rAdGT6

Fig. 1. Schema for generation of recombinant H-ras ribozyme containing adenovirus.The ribozyme sequence was subcloned into the Hindlll and Sail sites of pACCMVpL-pARS(+) to form the plasmid pACH-ras/RZ. The ribozyme expression shuttle vector(pACH-ras/RZ) and the adenoviral packaging plasmid pJM 17 were cotransfected into 293cells to generate the recombinant H-r«.vribozyme containing adenovirus.

previously (14). Expression of the cytoplasmic form of the anti-crbB sFv doesnot elicit any phenotypic alteration in erbB2 (+) or erbB2 (-) tumors of various

histológica! types (14). AdCMVLacZ, which encodes the Escherichia coliß-galactosidase, was graciously provided by Dr. R. Gerard (University ofTexas). Genomic DNA derived from the serotype 5 wild-type adenovirus

WT3IH) was used as a control for rAdGTo DNA analysis. WT300 adenoviruswas graciously provided by Dr. T. Shenk (Princeton University, Princeton,NJ ). Each adenovirus preparation was titered on the 293 cell monolayers usingplaque assay allowing direct determination of the PFU.

Adenoviral-mediated Gene Transfer. Subconfluently growing EJ cells

were infected with the recombinant adenovirus vectors by standard methodologies. Briefly, cells were overlaid with tissue culture medium containing 2%FBS and 500 PFU/cell of adenoviral vector added directly to the tissue culturewell. Cells were then incubated for 2 h at 37°Cin a 5% CO, atmosphere,

followed by replacement of low serum medium with complete medium described above. Incubations were then carried out for the indicated periods.

Analysis of Gene Expression. Total RNA was isolated from tumor cellsusing standard methods and quantified by spectrophotometric methods andelectrophoresis on agarose gel. Defined amounts of total RNA were loaded onnitrocellulose membranes and probed with a -'2P-labeled oligonucleotide, oli-gomer S'-GTTTCGTCCTCCAGGACTCAT-S', specific for H-ras ribozyme

sequences; a probe for the constitutive expressed gene PGK; or a probe fortargeted gene H-ras. The cDNA of PGK was graciously provided by Dr. J.Singer-Sam (City of Hope, Duarte. CA); the cDNA of H-ras was obtained

from American Type Culture Collection. The cDNA probes were labeled toachieve about 1 X IO9 cpm/f¿gby using a random primer system (Stratagene,

Menasha, WI). The oligomer probe was labeled by using terminal deoxynucle-otidyl transferase (GIBCO). Dot-blot hybridization was then performed usingQuikHybridization Solution (Stratagene) at 68°Cfor 1 h, the washing condi

tions and the development of the audioradiogram used conditions recommended by the manufacturer.

Animal Studies. 6-8-week-old athymic (nude) BALB/c mice were anesthetized, and adenoviral vector-infected EJ cells (IX IO1"cells/mouse) wereinjected s.c. in a volume of 100 /il of 1 X 107/ml. Cells had been infected

24 h prior to transplantation and were viable as assessed by trypan blueexclusion. The mice were maintained under standard pathogen-free conditions

and observed for evidence of tumor burden. Tumor volume was determined bythe formula: (length x widtrr)/2.

Results and Discussion

A recombinant adenovirus was constructed to encode the H-ras

ribozyme. The schema for this construction used standard methodsand is shown in Fig. 1. This methodology for recombinant adenovirusconstruction is based upon in vivo homologous recombination between the recombinant adenoviral shuttle vector and the adenoviralpackaging plasmid pJM17. In this strategy, recombinants allowingreplacement of the wild-type left terminus of the adenovirus genome,

with excision of the heterologous pBRX segments, may possesspackaging advantages compared to the oversized pJM17 adenoviralgenome-containing plasmid. The adenoviral shuttle vector containingthe H-ras ribozyme, pACH-ras/RZ was thus constructed and used to

derive the corresponding adenoviral vector. The resulting adenoviralvector would be predicted to contain the H-ras ribozyme expression

cassette inserted in place of deleted adenoviral El sequences (Fig.14). Based upon this genomic deletion, the resultant vector would bereplication-defective in non-ElA transcomplementing cells.

Cotransfection of the adenoviral shuttle vector pACH-ras/RZ and

the packaging plasmid pJM17 in the EIA transcomplementing cellline 293 resulted in the demonstration of multiple areas of cytopathiceffect consistent with adenoviral replication. Analysis of the viralDNA by PCR demonstrated the presence of a recombinant adenoviruspossessing characteristics consistent with the H-ra.c-expressing vectorpredicted in Fig. 1. Amplification of the pACH-ras/RZ shuttle vectorcontaining the H-ras ribozyme with the H-ra.v-specific primers

yielded a PCR fragment of 130 bp, consistent with the size of thepredicted H-ras insert (Fig. 2B). In Fig. 2B, lane 2, analysis of

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NEOPLASTIC REVERSION BY DELIVERY OF ANTl-ras RIBOZYME

rAdGTo genomic DNA yielded a similar band, again consistent withthe presence of the H-ras-containing recombinant adenoviral vector.Analysis for EIA sequences using the adenovirus ElA-specific prim

ers indicated the predicted PCR product of 1070 bp from DNA of theWT300 adenovirus. In contrast, this fragment was not derived inamplification of the rAdGTo, confirming the absence of EIA sequences in this recombinant adenovirus. The PCR analysis is thusconsistent with the derivation of an EIA(-), H-ras ribozyme-contain-ing recombinant adenovirus and also indicated that no wild-type virus

had been rescued by propagation.To verify the overall integrity of the adenoviral vector, restriction

analysis of genomic DNA of rAdGTo was performed (Fig. 2C).Analysis with a series of restriction endonucleases confirmed that the

(1.25) (9.2)EcoRICIal

ITR

(59.5) (76.0)BamHI EcoRI

I II ITR Ad5

10 2O 30 40 50 60 70 80 90 lOOm.u..

EcoRI Hindlll^11

H-ras ribozyme expression cassette

(kb)M 12345

0 6

0.1

E1 fragment

Ribozyme insert

EJ/mock

EJ/rAdGT6--48hrs

EJ/rAdGT6--72hrs

tRNA

Fig. 2. Analysis of H-ras containing recombinant adenoviral vector rAdGT6. A,structural map of rAdGT6 genomic DNA demonstrating localization of H-ras expression

cassette at site of E1 deletion. Restriction cndonuclease sites are indicated. The genomeis subdivided into 0-100 map units (m.u.). ITR indicates adenoviral inverted terminalrepeats of genomic termini. B, confirmation of rAdGT6 by PCR methods. Adenoviralvector genomic DNA was analyzed by primers specific for the H-ras ribozyme expressioncassette (PI and P2) or wild-type EIA gene (P3 and P4) analyzed by electrophoresis on1% agarose gel with cthidium bromide staining. Reactions for Lanes 1, 2, and J, withprimer PI and P2, and template DNA as follows: Lane I, pACH-ras/RZ plasmid DNA;

Lane 2, rAGT6 genomic DNA; Lane 3, no DNA. Reaction for Lanes 4, 5, ana 6 withprimer P3 and P4, and template DNA as follows: Lane 4, AdWT300 genomic DNA; Lane5, rAdGTo genomic DNA; Lane 6, no DNA. Markers on left demonstrate fragment sizein kb. The predicted size of the wild-type EIA fragment and the ribozyme insert areindicated. C, confirmation of rAdGTo by restriction analysis methods. Genomic DNAfrom rAdGTo was subject to digestion with the indicated restriction endonucleases.Products of the digestions were analyzed by electrophoresis on 1% agarose gel andethidium bromide staining. D, confirmation of H-ras ribozyme expression mediated byrAdGTo. The H-ras expressing adenovirus rAdGTo was used to infect the human bladder

carcinoma cell line EJ. Medium alone was mock infection control. At indicated timespostinfection, total cellular RNA was harvested from cells and blotted on a nitrocellulosemembrane with analysis for H-ras ribozyme containing transcripts using an H-ras ri-bozyme-specific '12P-labeled oligomer probe. As a negative control, yeast tRNA was also

blotted on the same membrane.

tt/ *°

A?

- H-ras

PGK

Fig. 3. Effect of rAdGTo on H-ras mRNA transcripts in EJ bladder cancer cells. Totalcellular RNA was isolated from EJ cells at various times after infection with either theribozyme-encoding adenovirus, rAdGTo, or the control adenovirus, AdCMVLacZ, and

blotted on nitrocellulose membranes. Analysis for specific transcripts was by hybridization analysis with 32P-labeled cDNA probes specific for either H-ras mRNA transcripts or

PGK mRNA transcripts.

overall configuration of the recombinant genome corresponded to thepredicted structure. To confirm the expression of the H-ras ribozyme,

the recombinant adenovirus rAdGTo was used to infect EJ cells invitro with subsequent derivation of total cellular RNA from the targetcells. Dot-blot analysis was then carried out using a radiolabeled oligoprobe specific for the encoded H-ras ribozyme sequences (Fig. 2D).

This study confirmed that only cells infected with rAdGTo werecapable of expressing the H-ras ribozyme. These studies thus confirm

that we have derived a recombinant adenovirus that is capable ofachieving expression of the H-ras ribozyme in tumor targets.

Previous studies from our lab have demonstrated that the H-rasribozyme is capable of sequence-specific cleavage of the GUC sequence in codon 12 of activated H-ras RNA (22-24).2 This sequence-

specific cleavage has been demonstrated in the FEMX-I human mel

anoma cell line and the EJ human bladder carcinoma cell lineexpressing the activated H-ras gene, as well as murine NIH3T3 cellstransfected with genomic DNA containing the transforming H-rasgene. These studies demonstrated the capacity of this sequence-

specific cleavage to accomplish inhibition of cellular proliferation andabrogation of tumorigenicity in stable clones derived for transfectionwith H-ras ribozyme-containing plasmid DNAs. We sought to eval

uate the hypothesis that highly efficient transient expression of theH-ra.v ribozyme mediated by the adenoviral vector could accomplish

a similar effect. In this regard, we have recently reported the efficacyof adenoviral-mediated gene transfer to human bladder epithelium.4

Using an E. coli ß-galactosidase (lacZ)-expressing recombinant ade

novirus, it could be demonstrated that >95% of a target cell population ofEJ cells could be successfully transfected in this manner. This H-ras-

transformed cell line thus served as a suitable target for this study.The EJ tumor targets were infected with the ribozyme-encoding

adenovirus rAdGTo, or AdCMVLacZ, an adenovirus encoding thereporter ß-galactosidase (LacZ), and were evaluated for the presence

of specific mRNA transcripts. In this analysis, it could be seen that theribozyme-encoding adenovirus induced a specific decrease in therelative levels of the H-ra.v transcript (Fig. 3). In contrast, infection of

the EJ cells with the control adenovirus AdCMVLacZ did not cause adecrease in the magnitude of the target H-ras mRNA transcript. The

4 C. Bass, G. Cabrera, A. Elgavish, B. Robert, G. Siegal, S. C. Anderson, D. C.

Maneval, and D. T. Curiel. Recombinant adenovirus-mediated gene transfer to genitourinary epithelium in vitro and in vivo, submitted for publication.

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NEOPLASTIC REVERSION BY DELIVERY OF ANTl-ras RIBOZYME

Days after infection

Fig. 4. Growth curve of the rAdGTò-infected EJ bladder cancer cells. The cells wereinoculated at a density of 5 X lO'Vwell in 35-mm dishes 24 h before infection and then infected

with rAdGTft or rAdGTl at 500 PFU/cell. Culture medium alone was used as mock infectioncontrol. Triplets of each treatment were counted daily from postinfection day 1 to day ft.

specificity of this effect was also demonstrated, as there was norelative decrement in the level of a control transcript corresponding tothe constitutively expressing gene PGK. Thus, the effect of rAdGTowas a highly specific down-regulation of the H-ras transcript.

The effect of adenoviral-mediated down-regulation of the H-ras tran

script on tumor cell growth was next evaluated. For this analysis, EJ cellswere infected with either the \\-ras ribozyme containing recombinant

adenovirus rAdGTo or the irrelevant recombinant adenovirus rAdGTl.Cells were infected at a multiplicity of infection of 500 PFU/cell. Preliminary studies had demonstrated that at this viral dose there is noappreciable nonspecific vector-associated cytotoxicity (data not shown).

In this study, cells infected with the irrelevant adenovirus rAdGTlexhibited growth kinetics similar to cells mock infected with media only(Fig. 4). In contrast, EJ cells infected with \\-ras ribozyme containing

adenovirus rAdGTo exhibited a significant suppression of growth. By 5days postinfection, viable cells could not be identified. The significantgrowth suppression was clearly a specific effect of the encoded ribozyme,because the adenoviral vector lacking this gene did not exert any significant effect on cell growth parameters.

The growth curve parameters observed in Fig. 4 indicated a time-dependent decrement in viable cell number induced by the ribozyme-

encoding adenovirus. This finding was consistent with the conceptthat the effects of ribozyme-encoded adenovirus eventuated in cell

death and not simply growth suppression. To further evaluate thisphenomenon, EJ cells were infected as before and evaluated directlyfor cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-

nyltetrazolium bromide assay. In this analysis, it could be seen thatinfection of the EJ cells with rAdGTo caused a time-dependent eradica

tion of tumor targets (Fig. 5). In contrast, infection of the EJ cells with thecontrol adenovirus AdCMVLacZ did not induce any significant cytocidaleffect. Increasing the dose of infecting adenovirus a further 5-fold (2500

PFU/cell) with the control adenovirus was still incapable of inducing acytocidal effect in these target cells (data not shown). Thus, the rAdGToadenovirus is capable of achieving a targeted cytocidal effect based uponthe encoded anti-ras ribozyme. At present, the mechanism of the cytoc

idal effect has not been fully characterized. Studies to evaluate thepossibility of an apoptotic mechanism are in progress.

A similar study was carried out to determine the effects of theadenovirus on EJ tumorigenicity. Heterotopic transplantation of the EJ

cells as xenografts in athymic nude mice resulted in the rapid development of progressive tumor nodules (Fig. 6). Infection of EJ with theirrelevant adenovirus rAdGTl followed by xenograft implantationresulted in a similar pattern of rapid tumor progression. Use of theH-ras ribozyme encoding adenovirus rAdGTo at the same multiplicity

of infection, however, resulted in complete abrogation of tumorigenicity. These results indicate that a population of tumor targets may betransduced at a sufficiently high efficacy to revert neoplastic progression in vivo.

The involvement of mutations in the ras gene family in the genesisof a variety of human neoplasms has made it an attractive target for

140 n

120-

= 100

80-

AdCMVLacZ

rAdGT6

Days after infection

Fig. 5. Effect of rAdGTo on viability of EJ bladder cancer cells. Cells were infectedwith rAdGTo, AdCMVLacZ, or culture media as a mock control. Cells were plated at500(1 cells/well in a %-well cluster dish and then, at various time points postinleclion.cells were analyzed for viability. Results are expressed as a percentage of cell viabilitycompared to mock controls. Experiments were performed 12 times each. Results representthe mean; bars, SD.

1600

1400 •

1200 •

1000 •

800 •

600

400 •

200 •

Days post implantation

Fig. 6. Effect of rAdGTo on tumorigenicity of EJ cells in athymic nude mice. EJ cellsinfected with rAdGTl or rAdGTfi at 500 PFU/cell were harvested 24 h posiinfection.Culture media alone was used as mock infection control. The treated EJ cells were injecteds.c. at 1 X IO*1cells/mouse. The temporal pattern of tumor volume increases was

monitored until 28 days postinjection. Each experimental group contained nine mice.

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mutation compensation gene therapy strategies. In this regard, rasgene mutations are present in approximately 90% of pancreatic ade-nocarcinomas, 45% of melanoma beyond Clark's level II, and 33% of

adenocarcinomas of the lung. The use of ribozymes as agents toachieve specific and targeted ablation of the transforming products ofthe raígene has been demonstrated in several contexts (20-27).2

Previous studies have demonstrated that a hammerhead ribozymetarget against the GUC sequence in codon 12 of H-ras can exert

profound effects on cell morphology, in vitro cell growth characteristics, and tumorigenicity of the human bladder carcinoma EJ. Specifically, ribozyme DNA cloned into an expression plasmid wastransfected into EJ cells, and the resulting transformants were shownto express decreased H-ras mRNA and p21 protein with profound

phenotypic alterations consistent with reversion of the neoplastictransformation. Similar findings have also been obtained in the context of human melanoma targets. These promising results have suggested the possibility of gene therapy interventions on this basis.

Effective implementation of ribozyme-based gene therapy strate

gies is contingent upon efficient delivery of the catalytic RNA to thetarget tumor cells. Strategies to accomplish intracellular delivery ofribozymes have been attempted using either exogenous delivery withnaked ribozymes or vector-based delivery to promote ribozyme ex

pression. In the case of exogenous delivery, the susceptibility of RNAoligonucleotides to ribonuclease attack has promoted the search formodifications to improve ribozyme stability while maintaining cleavage capability. While some of these chemical modifications canenhance intracellular stability, the utility of these modifications hasnot allowed in vivo use of naked RNA ribozymes. As an alternative,vector-based delivery systems have been explored. As noted, these

systems have allowed the derivation of clonal cell populations genetically modified to express the ribozyme construct. A vector systemcapable of high efficient transient gene expression has not beendescribed. Here we show that recombinant adenoviral vectors cansuccessfully accomplish efficient ribozyme delivery to tumor targetswith reversion of the neoplastic phenotype. This is the first descriptionof the use of this vector system for delivery of ribozyme constructs.The ability of recombinant adenoviral vectors to accomplish efficientin vivo transfection should allow the use of ribozyme-based gene

therapy strategies in the context of human neoplastic disease.

Acknowledgments

We thank Drs. Mark Kay and Jay Kolls for specific advice in the construction of the adenoviral vectors. We are also indebted to Connie Howton forexpert editorial assistance.

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1995;55:2024-2028. Cancer Res   M. Feng, G. Cabrera, Jessy Deshane, et al.   Adenoviral-mediated Delivery of an Anti-ras RibozymeNeoplastic Reversion Accomplished by High Efficiency

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