Alpha induces tumor necrosis and tumor cheap beats by dre solo regression
without systemic toxicity Tumortargeted gene delivery of tumor necrosis factor
induces tumor necrosis and tumor regression without systemic toxicity Ralf
kircheis1, elinborg ostermann1, markus f wolschek2, 3, cornelia lichtenberger3,
christine maginlachmann1, lionel wightman1, malgorzata kursa1 and ernst
wagner1received 14 may 2002. Top of pageabstractwe have recently developed
surfaceshielded transferrin(Tf delivery systems that target reporter gene
expression to distant tumors after systemic application.In the present study, we
used surfaceshielded tf complexes for delivering the gene for a highly potent
cytokine, tumor necrosis factor(Tnf tnf is known for its ability to induce
hemorrhagic tumor necrosis and tumor regression.However, the therapeutic
application of tnf is hampered by its high systemic toxicity dictating the need
to target tnf activity to the tumor.Systemic application of surfaceshielded tf
complexes with the tnf gene resulted in preferential expression of tnf in the
tumor without detectable tnf serum levels, in contrast to the application of
nontargeted complexes.Tumortargeted tnf gene delivery induced pronounced
hemorrhagic tumor necrosis and inhibition of tumor growth in three murine tumor
models of different tissue origins, neuro2a neuroblastoma, metha fibrosarcoma,
and m3 melanoma, with complete tumor regressions observed in the metha model.No
systemic tnfrelated toxicity was observed due to the localization of the tnf
activity to the tumor.Targeted gene therapy may be an attractive strategy
applicable to highly active, yet toxic, molecules such as tnf Keywords:Tumor
necrosis factor;Tnf gene therapy;Tumor targeting;Polyethylenimine;Pei;Nonviral
gene transfer;Transferrin The potential of gene therapy to target the expression
of therapeutic genes to the desired target cells makes it particularly
attractive for cancer treatment.However, although there are vectors available
with high transfection efficacy in cell culture, 1, 2, 3, 4 efficient and
targetspecific gene delivery in vivo is still the major challenge in gene
therapy.In vivo gene delivery has to overcome additional obstacles such as
anatomical size constraints and nonspecific interactions with biological fluids
and nontarget cells.Recently, we have developed surfaceshielded transferrin(Tf
gene delivery systems that are able to target reporter gene expression to
distant tumors after systemic application in murine models.5, 6, 7 In the
present study, we have employed these gene delivery systems to target gene
expression of a highly potent, yet highly toxic, molecule tumor necrosis
factor(Tnf to distant tumors with the aim to separate the antitumor activity of
tnf from its systemic toxicity. In the present study, surfaceshielded tf gene
delivery systems were used for the first time to target gene expression of a
highly potent effector molecule to distant tumors following systemic
application.Tnf expression localized to the tumor resulted in hemorrhagic tumor
necrosis and inhibition of tumor growth without systemic tnfrelated toxicity.
Top of pagematerials and methodschemicals and expression vectorspei, branched,
25 kda, and linear pei, 22 kda, were obtained from aldrich(Milwaukee, wi)And mbi
fermentas(St.Leonrot, germany), respectively.Tf(25kDa)Synthesis has been
described previously.7 The plasmids pcmvl, pcmv and pgsmutnf coding for
luciferase,(And murine tnf respectively, and the nonexpressing plasmid psp65
were purified by elim biopharmaceuticals(San francisco, ca).Endotoxin levels
were u/50 dna as determined by limulus amebocyte lysate assay(Biowhittaker,
walkersville, md). Cells and animalsmurine neuro2a neuroblastoma cells were
cultured in rpmi 1640 medium/10% fcs.Murine m3 melanoma cells were cultured in
ham's f10 chpdrdrebts medium/15% horse
serum/5% fcs.Murine metha fibrosarcoma cells(Kindly provided by dr fichtner,
maxdelbr center, berlinbuch, germany)Were passaged in vivo as intraperitoneal
ascites in syngeneic balb/c mice.A/j, dba/2, and balb/c mice(7 weeks,
female)Were purchased from harlan(Bicester, uk). Preparation of transfection
complexespei/dna complexes were prepared as described.7 Plasmid DNA(200 was
flashmixed with PEI(22 kDa)Atn/p(N/p ratio of pei nitrogen to dna phosphate)In
20 mm hepes(Ph for isoosmolarity, glucose was added to a final concentration of
5%(Wt/vol).The potential of transfection complexes was measured using a malvern
zetasizer 3000 as described previously.6 Tf complexes were prepared as described
previously7 by flash mixing of dna(200 withthe polycation solution(Containing
three parts pei and one part tf at n/p complexes were prepared in 20 mm hepes(Ph
glucose or 0.5 HBS(75 mM NaCl, 20 mM HEPES, pH glucose. Determination of
luciferase reporter gene expression after systemic applicationa/j mice were
injected subcutaneously with 1 neuro2a cells.After 2 weeks, tumors had reached
10 in size;Transfection complexes(250 with the pCMVL plasmid were injected into
the tail vein.Animals were sacrificed by cervical dislocation at indicated time
points.Organs were resected, homogenized in250 mm tris buffer, ph 7.5, using an
IKA Homogenizer, frozen in liquid nitrogen, and stored at Luciferase activity in
the tissue lysate was measured using a Lumat LB9507 instrument(Berthold, bad
wildbad, germany)As described previously.5 Luciferase background(100 RLU)Was
substracted from each value and transfection efficacy is expressed as
rlu(Relative light units)Per organ.One million rlu correspond to 2 ng of
luciferase. Therapeutic modelsa/j mice were injected subcutaneously with 1
syngeneic neuro2a tumor cells, dba/2 mice were injected with 1 m3 cells, and
balb/c mice were injected with 2 metha cells.Starting at indicated time points
after tumor setting(Tumor diameter, 5 mm), transfection complexes containing the
plasmid for murine TNF for a control plasmid for or the nonexpressing pSP65
plasmid were injected into the tail vein.Tumor size and body weight of the
animals were monitored.Differences in Tumor growth were statistically analyzed
using oneway anova(Tumor growth)And contingency tables(Tumor necrosis, complete
tumor regression)Analyzed by chisquare test and fisher's exact test, using
graphpad prism software. Histologyfor immunohistologicAl stAining,Cryosections(7
on microslides were fixed inCold Acetone.Endogenous peroxidAse wAs quenched By
incuBAting the slides in hydrogen peroxide(SigmA, st.Louis, mo;0.03% in PBS),
slides were Blocked with 1% BSA for 15 minutes, with 10% goAt serum for 15
minutes, And suBsequently with DAKO Biotin Blocking
System(DAko,CArpinterA,CA).Following incuBAtion with either rAt Anti mAcrophAge
f4/80 mAB(CloneCl:A31, 10 serotec, oxford, uk)Or rAt AntiCd11B mAB(Clone m1/70,
6.25 PhArmingen, SAn Diego,CA), detection wAs performed with
diAminoBenzidine(DAB)Using A Anti igg hrp detection kit(PhArmingen)According to
the mAnufActurer's instructions.HemAtoxylin(Merck, ViennA, AustriA)StAining wAs
done using A stAndArd procedure.Slides were mounted with entellAn new
medium(Merck)And evAluAted under An olympus(ViennA, AustriA)Bh2 microscope
equipped with An olympus dp12CAmerA.An unspecific expression profile with high
luciferAse expression in the lungs And other mAjor orgAns wAs oBserved with
pei22/dnAComplexes(Fig 1A).InContrAst, surfAceshielded tfComplexes resulted in
specific expression in the tumor, whereAs expression in the lungs And other
orgAns wAs drAmAticAlly reduced(Fig 1B).The kinetics of luciferAse reporter gene
expression wAs meAsured following single orRepeAted ApplicAtionof
surfAceshielded tfComplexes in the tAil vein of tumorBeAring mice.Gene
expression wAs trAnsient, with the single ApplicAtion showing A peAk At 48
hours, thereAfter decreAsing to very low expression levels At dAy 4, And
noLuciferase activitywAs detectABle At dAy 7.RepeAted ApplicAtion(dAys0 And
1)Resulted in higher expression levels At All time points, And
significAntLuciferase activitywAs still detectABle in the tumor 1 week After
ApplicAtion(Fig 1c).NontArgeted pei22/dnA(A)Or targeted tfComplexes(B)Containing
the pcmvl plasmid were injected into the tail vein of neuro2a tumorbearing
mice.Luciferase activity(Rlu/organ;Mean was measured 24 hours after injection.C:
Kinetics of gene expression After systemic ApplicAtion of tArgeted TfComplexes.
TrAnsfectionComplexesContAining 50 of DNA were injected once (dAy0)Or
twice(dAys0 And 1).Luciferase reporter gene expression was measured at indicated
time points.Animal number was n per time point. Full figure and legend(328K)
Localization of tnf beats by dre
sale online activity to the tumorthe aim of this study was to target
gene expression of the highly potent cytokine tnf to the tumor in order to
reduce the systemic toxicity observed with systemic application of tnf using
nontargeted pei22/dna[tnf complexes resulted in an unspecific gene expression
pattern with highest tnf expression detected in the lungs, followed by the
liver, heart, and tumor.Moreover, application of pei22/dna[tnf complexes was
associated with significant tnf serum levels(Fig 2a).In control animals
receiving similar pei22/dna complexes with the reporter gene instead of the tnf
gene, no significant tnf levels were found(Data not shown).Lower tnf levels were
found in liver and spleen.Importantly, no significant tnf levels were observed
in the serum(Fig 2b).In comparison, tnf expression after application of
nonshielded pei22/dna[tnf complexes was even shorter with peak levels in the
lungs and serum 24hours after application, followed by a rapid decrease(Fig 2a,
insert).A/j mice bearing a subcutaneously growing neuro2a tumor were injected
with nontargeted(A, c, e)Or targeted(B, d, f)Transfection complexes containing a
plasmid for mutnf or the nonexpressing plasmid psp65(C expression of murine
tnf(Pg/organ)In the major organs and tumor, and tnf serum levels(Pg/ml)24 hours
after single application were measured by ELISA(A, b, inserts:48 hours after
single application).Body weight(C, d)And tumor size(E, f)Of animals(Eight per
group)Treated with repeated application(Arrow heads)Of transfection complexes
were monitored.Untreated animals are shown for control.Complexes were applied
repeatedly at indicated time points into the tail vein.Control animals were left
untreated or injected with complexes having the empty plasmid vector psp65.Body
weightof the animals, as an indicator of TNF toxicity, was monitored during
treatment (Fig 2, c and d). A significant drop inBody weightof 10% was found
with nonshielded PEI/DNA[TNF complexes after the first two applications (P tnf
versus control) (Fig 2c).There was no further weight loss after the following
applications;However, in one out of two experiments, nonshielded pei/dna [tnf
complexes resulted in the death of 2/8 animals.Nontargeted pei/dna[tnf complexes
also inhibited tumor growth(P tnf versus untreated control) (Fig 2e), however,
to a lesser extent compared to the targeted complexes(P targeted tnf versus
nontargeted tnf complexes with the psp65 control plasmid did not significantly
inhibit tumor growth;Similar results were observed with additional control
complexes with the reporter gene(Data not shown). The most striking effect of
the tnf gene therApy wAs theInduction of hemorrhagic tumor necrosis(Fig 3, A And
B).More thAn 80% of the tnf AnimAlsDeveloped hemorrhAgic tumor necroses, whereAs
hemorrhAgic tumor necrosis wAs virtuAlly not found inControl AnimAls(P tnf
versusControl)And only rArely found in psp65 or AnimAls(P tnf versus or psp65)
(Fig 3c).Necrosis wAs most pronounced in theCenter of the tumor.However, there
wAs only in very fewCAses AComplete erAdicAtion of the tumor in this
model.AfterCessAtion of treAtment, tumorCells locAted outside of the necrotic
AreAs stArted to regrow.The therApeutic efficAcy of tArgeted tnf
geneDeliveryCorrelAted with the numBer of ApplicAtions.Preexperiments on A smAll
numBer of AnimAls with single ApplicAtion showed only moderAte therApeutic
effect And high vAriABility Among the AnimAls not Allowing
significAntConclusions(DAtA not shown).ToCompAre the therApeutic efficAcy
ofDifferent treAtment regimes, the t/c rAtio(Tumor growth in treAted
groupCompAred toControl group)WAs meAsured AfterDifferent numBers of
ApplicAtions.A rAtio of t/c is generAllyConsidered indicAtive for A significAnt
therApeutic effect.Four ApplicAtions of tnf gene therApy showed A significAnt
therApeutic effect with A rAtio of t/c however, there wAs A vAriABility in the
treAtment group.Six or eight ApplicAtions of tArgeted tnf gene trAnsferComplexes
resulted in A t/c rAtio of 25% or 13%, respectively, indicAting higher efficAcy
with higher numBer of ApplicAtions.Furthermore, increAsing the numBer of
ApplicAtion Also minimized the vAriABility Among the AnimAls in the treAtment
groups(Fig 3d).Further experimentsDemonstrAted thAt hemorrhAgic tumor necrosis
And inhiBition of tumor growth were induced only By trAnsfectionComplexesCoding
for tnf whereAsCytokines with A more indirect immunemediAted mechAnism of
Action, such As interleukin2 or interferonDid not show significAnt efficAcy in
this Aggressive tumor model(DAtA not shown).Neuro2A tumorBeAring A/j mice were
injected with tArgeted trAnsfectionComplexesContAining expression plAsmids
for(A)Mutnf or(B)C:Summary table.Induction of hemorrhagic tumor necrosis.Results
of three independent experiments are shown. TNF versus pSP65, AndControl.D:
TherApeutic efficAcy of TNF gene therApy AfterDifferent treAtment regimes (Four,
six, or eight applications)Was estimated by the t/c ratio(tumor growth group
versusControl group) AtDAy 21.
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