249 long term transgene expression from high capacity adenoviral vectors delivered to fetal muscle in utero

249 Long Term Transgene Expression from High Capacity Adenoviral Vectors Delivered to Fetal Muscle In Utero Molecular Therapy �������� ��� ���� ���������������� �������� ���� ������© ����������� �!���[.]

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248 Correction of the Dystrophin Gene

Mutation in the mdx5cv Mouse Model of Duchenne

Muscular Dystrophy Mediated by Chimeric and

DNA Oligonucleotides In Vitro and In Vivo

Carmen Bertoni,1 Thomas A Rando.1

1 Department of Neurology, Stanford University Medical Center,

Stanford, CA, United States.

Gene correction represents an appealing option for the treatment

of genetic disorders due to the prospect of permanent restoration of

gene expression We have investigated the possibility of inducing

single base pair alterations at the genomic level to restore the

expression of dystrophin in mouse models of Duchenne muscular

dystrophy, a severe muscle disease caused by mutations in the

dystrophin gene We have shown the ability of chimeric RNA/DNA

oligonucleotides (chimeraplasts) to correct a point mutation in the

dystrophin gene in the mdx mouse We have also shown the feasibility

of using chimeraplasts to mutate a base in an intron/exon boundary

of the dystrophin gene to alter splicing, an approach that may be

applicable to a variety of dystrophin gene defects We have now

compared the correction ability of chimeraplasts to that of DNA

oligonucleotides in muscle cells of the mdx 5cv mouse This model has

a point mutation in exon 10 of the dystrophin gene that creates a

cryptic splice site Exon 10 is thus aberrantly spliced resulting in

alteration of the dystrophin coding sequence leading to a lack of

dystrophin expression

We have designed a targeting chimeraplast (MDX7¹) and targeting

DNA oligonucleotides (MDX7² and MDX7³) to specifically correct

the mdx 5cv mutation Each oligonucleotide is perfectly homologous

to the region of exon 10 of the mdx 5cv dystrophin gene containing the

mutation, except for a mismatch at the mutated base The chimeric

oligonucleotide is designed to pair with both strands of the DNA;

the DNA oligonucleotides are designed to pair with either the coding

(MDX7³) or the non-coding strand (MDX7²) As controls, we have

used a chimeraplast (MDX8¹) and DNA oligonucleotides (MDX8²

and MDX8³) identical to the targeting oligonucleotides but lacking

the mismatch with the mdx 5cv mutation

Fluorescently labeled oligonucleotides are efficiently taken up in

muscle precursor cells in vitro using all 3 types of oligonucleotides.

Fluorescence persists longer in cells transfected with MDX7² or

MDX7³, suggesting that DNA oligonucleotides have increased

stability compared to chimeraplasts Restoration of dystrophin

expression was assessed at the mRNA and protein level All targeting

oligonucleotides were capable of restoring dystrophin expression,

while control oligonucleotides had no effect Gene correction was

demonstrated at the genomic level in cells transfected with targeting

oligonucleotides Quantitative RT-PCR indicated that the level of

gene correction varied between 0.2 to 5% The most efficient

oligonucleotides were the chimeraplast (MDX7¹) and the DNAoligonucleotide that was designed to anneal with the coding strand(MDX7³)

The chimeric and DNA oligonucleotides also corrected the mdx 5cv

mutation in vivo as determined by the restoration of dystrophin

expression The expression of dystrophin was assessed as early as

2 weeks after injection and was stable for at least 3 months afterinjection

Our studies provide evidence that oligonucleotide-mediated genecorrection is a feasible approach to the treatment of certain geneticdisorders in which long-term gene expression is required Thus thistechnology has the potential to be a viable, non-viral approach tostable restoration of gene expression

249 Long-Term Transgene Expression from High-Capacity Adenoviral Vectors Delivered to Fetal Muscle In Utero

Roberto Bilbao,1 Daniel Reay,1 Volker Biermann,2 ChristophVolpers,2 Zhilong Jiang,1 Stefan Kochanek,2 Paula R Clemens.1,3

1 Neurology, University of Pittsburgh, Pittsburgh, PA; 2 Center for Molecular Medicine, University of Cologne, Cologne, Germany;

3 Department of Veterans Affairs Medical Center, Pittsburgh, PA.

In utero gene delivery holds promise for the treatment of

hereditary diseases such as Duchenne muscular dystrophy (DMD)

To date, efficient transduction has been achieved using generation adenoviral vectors Due to the large size of the dystrophingene cDNA (14 kD), gene transfer of the full-length cDNA willrequire a vector with a larger insert capacity such as the high-capacityadenoviral (HC-Ad) vector In this study, we analyzed the longevity

first-of transgene expression achieved by direct HC-Ad vector-mediated

gene delivery to muscle in utero We also studied the efficiency of

muscle gene delivery by HC-Ad vectors with intravascular delivery

We first evaluated the transduction levels in muscle afterintramuscular delivery of an HC-Ad vector carrying the lacZ gene(AdGS46) to fetal C57BL/6 mice 16 days after conception (E-16).Hind limb muscles were collected 1 and 5 months after infection and

transgene expression in muscle were found We also investigatedintravascular delivery of HC-Ad vector to C57BL/6 E-16 fetusesand observed high transduction efficiency in limb muscles In addition,higher survival rates were observed in those mice transduced with

an HC-Ad vector as compared to a first-generation Ad vector Toassess the potential of HC-Ad vector-mediated gene transfer tofetal muscle in a therapeutic model, we performed intramuscularinjections of an HC-Ad vector carrying the dystrophin gene (AdDYS)

to E-16 mdx mice, the animal model for DMD Immunohistochemical staining showed dystrophin expression in muscle of mdx mice transduced in utero with AdDYS.

Our results demonstrate that 1) Long-term transgene expressioncan be achieved by HC-Ad vector-mediated gene delivery to fetalmuscle; 2) The HC-Ad vector can deliver full-length dystrophin to

fetal muscle in utero.

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250 A Novel Approach To Identify Patients

with Duchenne Muscular Dystrophy Caused by

Stop Codon Mutations Using Aminoglycoside

Antibiotics

Shigemi Kimura,1 Tishihiko Miyagi,1 Takashi Hiranuma,1

Kowashi Yoshioka,1 Shiro Ozasa,1 Kaori Ito,1 Makoto

Matsukura,1 Makoto Ikezawa,1 Masafumi Matsuo,2 Yasuhiro

Takeshima,3 Teruhisa Miike.1

1 Department of Child Development, Kumamoto University School

of Medicine, Kumamoto, Kumamoto, Japan; 2 Division of

Molecular Medicine, Kobe University Graduate School of Kobe,

Kobe, Hyogo, Japan; 3 Department of Pediatrics, Kobe University

Graduate School of Kobe, Kobe, Hyogo, Japan.

Intro: Aminoglycoside antibiotics have been found to suppress

premature stop codons located in the defective dystophin gene in

mdx mice, suggesting a possible treatment for Duchenne muscular

dystrophy (DMD) However, it is very difficult to find patients

that are applicable for this therapy, because: 1) only 5 to 10% of

DMD patients have nonsense mutations in the dystrophin gene, 2)

it is challenging to find nonsense mutations in the gene because

dystrophin cDNA is very long (14kb), and 3) the efficiency of

aminoglycoside-induced read-through is dependent on the type of

nonsense mutation Recently, our research has focused on MyoD, a

transcriptional factor that has the ability to differentiate fibroblasts

into myotubes in vitro Adenoviral vectors encoding MyoD,

regulated by CAG Promoter (AdMyoD), can efficiently transduce

fibroblasts to express MyoD In this study, we introduce an easy

system to identify patients for this therapy and report for the first

time, that dystrophin expression was detected in myotubes of DMD

patients using gentamicin

Methods:Fibroblasts were isolated from six DMD patients In

patient 1, a deletion of exons 48~50 in the dystrophin gene resulted

in an out of frame pattern of the gene Patients 2-6 had nonsense

mutations in the dystrophin gene; the stop codon is TGA for patients

2-4 and TAA for patients 5 and 6 Control fibroblasts were isolated

from a non-DMD patient The fibroblasts were infected in vitro

with AdMyoD using a multiplicity of infection (MOI) of 100

Following infection, the cells were cultured in DMEM supplemented

with 2% FBS and 300 mg/ml of gentamicin At 2 weeks

post-infection, the dystrophin expression was analyzed by dystrophin

staining and Western blotting analysis

Results:The in vitro immunofluorescence staining and Western

blot analysis for dystrophin showed that dystrophin expression

was not detected in the myotubes of patient 1 (deletion of

dystrophin gene) cultured with and without gentamicin In contrast,

dystrophin expression was detected in myotubes of patients 2, 3

and 4 (stop codon mutation TGA) cultured with gentamicin, but

not detected in myotubes cultured without genatamicin Interestingly,

dystrophin expression was not observed in myotubes from patients

5 and 6 with the stop codon mutation TAA, in spite of being cultured

with gentamicin Dystrophin expression was detected in control

myotubes from fibroblasts of a non-DMD patient after culturing

them with and without gentamicin

Discussion:We have developed a system to identify DMD patients

caused by stop codon mutations in the dystrophin gene that are

eligible for gentamicin treatment By monitoring dystrophin

expression of myotubes differentiated from fibroblasts infected with

AdMyoD and cultured in gentamicin, we are able to determine which

patients will benefit from such treatments In addition, our results

show that this system for the aminoglycoside treatment is far more

effective for DMD patients that have nonsense mutation TGA than

for patients that have nonsense mutation TAA

251 Prolonged Dystrophin Expression and Functional Correction of mdx Mouse Muscle Following Gene Transfer with a Helper- Dependent (Gutted) Adenovirus Encoding Murine Dystrophin

Renald Gilbert,1 Roy W R Dudley,2 An-Bang Liu,3 Basil J.Petrof,2 Josephine Nalbantoglu,4 George Karpati.4

1 Genomics and Gene Therapy Vector Group, Biotechnology Research Institute, NRC, Montreal, QC, Canada; 2 Respiratory Division, McGill University, Montreal, QC, Canada; 3 Department

of Neurology, Tzu Chi Medical Center, Hualien, Taiwan;

4 Neuromuscular Research Group, Montreal Neurological Institute, Montreal, QC, Canada.

Dystrophin gene transfer using helper-dependent adenoviruses(HDAd), which are deleted of all viral genes, is a promising option

to treat muscles in Duchenne muscular dystrophy We investigatedthe benefits of this approach by injecting the tibialis anterior (TA)muscle of neonatal and adult dystrophin-deficient (mdx) mice with

a fully deleted HDAd (HDCBDysM) This vector encoded twofull-length murine dystrophin cDNAs regulated by the powerful

post-injection of neonatal muscles, 712 fibers (42% of the total number

of TA fibers) were dystrophin positive (dys+), a value that did notdecrease for 6 months (the study duration) In treated adults, maximaltransduction occurred at 30 days post-injection (414 dys+ fibers,24% of the total number of TA fibers), but decreased by 51% after

6 months All studied aspects of the pathology were improved inneonatally-treated muscles: the percentage of dys+ fibers withcentrally localized myonuclei remained low, localization of thedystrophin associated protein complex was restored at the plasmamembrane, muscle hypertrophy was reduced, maximal forcegenerating-capacity and resistance to contraction-induced injurieswere increased The same pathological aspects were improved inthe treated adults, except for reduction of muscle hypertrophy andmaximal force generating capacity We demonstrated a strong humoralresponse against murine dystrophin in both animal groups, but mildinflammatory response occurred only in the treated adults Ourdata indicate that HDCBDysM is one of the most promising andefficient vectors for treating DMD by gene therapy, and that earlymuscle treatment using this vector would mitigate the DMDpathology more efficiently

252 Gel-Based Delivery of Recombinant AAV Vectors to Adult Murine Diaphragm

Thomas J Fraites, Jr.,1 Cathryn Mah,1 Irene Zolotukhin,1 Barry

We constructed rAAV vectors based on rAAV serotypes 1, 2, and

5, and evaluated their utility for diaphragmatic gene delivery withand without a gel-based delivery vehicle Recombinant AAV2 vectorplasmids encoding the cytomegalovirus immediate-early promoter-driven beta galactosidase reporter gene were cross-packaged into

AAV1, 2, and 5 capsids as previously described (Zolotukhin, et al.,

2002 Methods 28(2):158-67) Vectors were mixed at room

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temperature with a water-soluble, bacteriostatic, gelatin-based gel

and directly applied to the abdominal surface of the diaphragm Free

virus, without vehicle, was also directly applied to control

particles Four weeks after gene delivery, diaphragm tissues were

harvested and assessed for enzyme activity by X-gal staining

B.J.B and the University of Florida may be entitled to patent

royalties for inventions described in this abstract

253 Widespread Gene Expression of

d-sarcoglycan in the Bio14.6 Dystrophic Hamster

Hanidleg Muscles by Pressurized Delivery of a

Double-Stranded AAV Vector

Tong Zhu,1 Liqiao Zhou,1 Bing Wang,1 Juan Li,1 Xiao Xiao.1

1 Molecular Genetics and Biochemistry, University of Pittsburgh,

Pittsburgh, PA.

Introduction: Gene transfer of the missing d-sarcoglycan in the

limb girdle muscular dystrophy hamster Bio14.6 by AAV vectors is

an effective treatment But systemic gene delivery through blood

vessel and transduction efficiency of AAV vectors need further

improvement Here we tested the pressurized intra-arterial injection

method to transfer a novel double-stranded AAV vector carrying the

d-sarcoglycan gene into the hindlimb muscle of Bio14.6 hamsters

Method: To deliver the AAV vector through artery into a large

group of the muscles, the femoral vessels were carefully dissected

under a surgical microscope Two overlapping rubber tourniquets

were transmuscularly placed at the level of the proximal thigh A

microvascular clamp was placed to temporarily occlude the femoral

vessels A 32G intracranial catheter was canulated distally into the

femoral artery After the tourniquets were tightened , 4x1012 particles

of dsAAV-CMV-d-sarcoglycan diluted in 1 ml PBS was injected

into the artery as fast as possible (normally in 8-10s) Local

Intramuscular injection was also performed as a positive control

with 2x1012 AAV vector particles into the gastrocnemius and tibialis

anterior muscles, respectively All animals were sacrificed 1 or 2

months after injection Cryosections of Quadriceps, GAS and TA

muscles were obtained for anit-d-sarcoglycan immunostaining

Results: The untreated muscle cells showed a degeneration

morphology without sarcoglycan expression; In contrast, in the

intravascularly treated hindlimb, sustained and uniform expression

of sarcoglycan was observed on the cell membranes of all muscle

groups downstream of the vasculature However, in local

intramuscular injection group, the sarcoglycan only expressed locally

on the membrane of muscle cells in the injected muscle Uneven

expression of the d-sarcoglycan was observed In general, no toxicity

was observed for both gene delivery methods

Conclusion: The pressurized intravascular injection method can

systemically deliver the dsAAV-CMV-d-sarcoglycan to the hindlimb

muscles, which offers potential clinical significance for future gene

of dystrophic mdx mouse muscle However, viral vector mediated

delivery of dystrophin to all muscle fibers remains a challenginggoal Insulin-like growth factor-I (Igf-I) was found to enhance muscleregeneration and to maintain muscle mass and function in old anddystrophic animals Igf-I is a secreted polypeptide and can thereforetarget virus infected and non-infected cells by binding to its receptorand triggering proliferative and differentiation responses and anti-apoptotic pathways Our goal is to co-deliver dystrophin and Igf-

I to dystrophic muscle to determine if the protective effect of Igf-I

is synergistic with the beneficial effects of dystrophin in ameliorating

the mdx phenotype We have cloned and characterized the isoforms

of Igf-I that are expressed in normal and dystrophic mouse muscle.Based on this information, we have generated several AAV vectorsthat express the major muscle isoform of Igf-I Our AAV vectorsexpress Igf-I under the control of the CMV and the muscle specificpromoters, CK6 and desmin We have developed a real time PCRassay to quantify RNA expression and to characterize the relativestrength of the promoters Intramuscular injection of these AAVIgf-I vectors into C57/BL10 mice leads to levels of Igf-I mRNAexpression up to 500-fold above normal Currently, we areinvestigating the effects of this overexpression on the muscle functionand morphology Additionally, we have co-injected AAV carryingmicro-dystrophin with AAV carrying Igf-I into dystrophic muscleand are studying their relative and combined potential for reversing

the dystrophic pathology of the mdx mouse.

255 Local Delivery of VEGF165 by AAV Vectors Protects Skeletal Muscle from Injury and

Promotes Muscle Regeneration

Nikola Arsic,1 Serena Zacchigna,1 Lorena Zentilin,1 GenaroRamirez-Correa,1 Sabrina Tafuro,1 Lucia Pattarini,1 AlessandroSalvi,2 Gianfranco Sinagra,2 Mauro Giacca.1

1 Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy; 2 Cardiology Unit, Ospedale Maggiore, Trieste, Italy.

Vascular endothelial growth factor (VEGF) is a main regulator ofblood vessel formation during embryogenesis and a potent inducer

of neovascularization during adult life Recent evidence suggeststhat VEGF activity is not strictly specific for endothelial cells, but

is also exerted on other cell types

Here, we report on the role of VEGF165 in promoting myogenicprecursor cell differentiation to form multinucleated myotubes invitro, as well as in the enhancement of muscle regeneration in vivo

By immunofluorescence on cultured satellite cell-derived primarymyoblasts, we showed that VEGFR2 is strongly upregulated after

2 days of culture under differentiating conditions, and remains highlyexpressed until the last stages of the differentiation process Similarresults were obtained using the C2C12 myogenic cell line

In both primary myoblasts and C2C12 cells VEGF determinedcell cycle arrest and protected cells from apoptotic death Moreover,the administration of recombinant VEGF during C2C12differentiation resulted in a significant increase in the number and inthe length of the newly formed myotubes, resulting from myoblastfusion

The effects of VEGF on muscle cell survival and regeneration invivo were assessed by the injection of a high titer preparation of an

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AAV vector expressing VEGF in two established murine models of

muscle damage Muscle fiber injury was obtained by injection of

either 50% glycerol or 1 mM cardiotoxin, which both induce rapid

destruction of muscle fibers and strong inflammatory reaction The

former treatment also recapitulates some of the hallmarks that define

the physiopathology of Duchenne muscular dystrophy

By immunohistochemistry we observed that injury with both

glycerol and cardiotoxin induced expression of VEGFR2 in muscle

fibers Delivery of AAV-VEGF resulted in a remarkable improvement

in the preservation of viable fibers and in the induction of fiber

regeneration at day 20 after damage with both agents Preservation

of tissue architecture was almost complete after injury with glycerol

This effect involved a marked reduction in fiber apoptosis (as detected

by reactivity to anti-caspase-3 antibody) and an increase in the

number of regenerating myofibers Activity of AAV-VEGF strictly

correlated with the dose of vector administered

These results demonstrate that VEGF exerts a powerful and

specific effect on muscle cell survival and myogenic differentiation

This conclusion implicates that gene delivery of VEGF, besides

induction of therapeutic angiogenesis, might be considered for the

induction of muscle regeneration for the treatment of a variety of

muscular disorders

256 Rapid Identification of Novel Canine

Models of Duchenne Muscular Dystrophy

Bruce F Smith,1 Glenn E Morris,2 Joe N Kornegay,3 Richard J

Bartlett.4

1 Scott-Ritchey Research Center, Auburn University, Auburn, AL,

United States; 2 NEWI, University of Wales, Wrexham, United

Kingdom; 3 College of Veterinary Medicine, University of

Missouri, Columbia, MO, United States; 4 NIAMS, National

Institutes of Health, Bethesda, MD, United States.

Duchenne muscular dystrophy (DMD) is an X-linked,

progressive muscle wasting disease with fatal consequences, which

is caused by mutations in the human dystrophin gene DMD

presents unique challenges to gene therapy, due to the size of the

gene and resulting cDNA and the wide variety and complexity of

the mutations involved As an X-linked recessive disease, new

mutations present themselves at higher rates than in autosomally

inherited diseases In addition, mutations in DMD differ from those

classically seen in inherited disease, with an emphasis on deletions

Animal models for DMD have been described in mice, cats and dogs

with mutations in the respective dystrophin genes, located on the

x-chromosome Both cats and mice have less severe forms of the

disease In the murine model, additional mutations such as a utrophin

gene knockout must be bred into the mdx background to reproduce

the pathology, symptomatology and fatal consequences found in

DMD patients Only in dogs does the disease carry the full spectrum

of clinical phenotypes found in patients with DMD, including the

fatal consequences of muscle wasting Thus, only canine

dystrophinopathies recapitulate the human disease with a single

gene mutation In dogs and humans the structure, size and exon

junction location of the dystrophin genes are remarkably similar

Thus, it is not surprising that the mutations in the animal models

described thus far prove to be different mutations Any therapy

developed for treatment of this spectrum of mutations must be

capable of overcoming the consequential spectrum of clinical

conditions To maximize the potential for pre-clinical evaluation of

potential therapies, a spectrum of mutations in canine breeds with

the consequential continuum of clinical phenotypes would prove

invaluable To date, canine models have been described in the Golden

Retriever, Rottweiler, and German Short Haired Pointer breeds

We have developed a program to rapidly screen possible new

canine models of dystrophin deficiency using a combination of

specific antibodies and rapid, directed sequencing In the initial

screen, antibody binding data is used to identify the likely portion

of the molecule affected by the mutation This region is then amplified

by RT-PCR in one kilobase sections and sequenced Data fromthese PCR reactions also provides an indication of small deletions

or insertions, either due to the failure of the PCR reaction to amplify,

or due to alterations in amplicon size Analysis of the cDNAsequence data allows the specific mutation to be identified or it mayindicate possible genomic alterations that affect cDNA structure.This approach allows rapid identification of the mutation in themodel, allowing the suitability of a model to be determined withoutsignificant investment or delay By determining the mutation inadditional dogs that have presented with clinical muscular dystrophy,

a spectrum of single gene mutations in canine models can be describedthat will allow more appropriate screening of therapeutic approachesthan is possible in inbred mice Data from the analysis of newcanine models of Duchenne Muscular Dystrophy will be presented

257 Muscle Stem Cells Promote Nerve Regeneration and Contribute to the Development

of Neuronal Tissues

Zhuqing Qu-Petersen,1 James Cummins,1 Aiping Lu,1 ArvydasUsas,1 Ron Jankowski,1 Makoto Ikezawa,1 Ryosuke Kuroda,1

William de Groat,2 Johnny Huard.1

1 Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA;

2 Pharmacology, University of Pittsburgh, Pittsburgh, PA.

Muscle-derived stem cells (MDSC) isolated from normal neonatalmouse skeletal muscle via the preplate technique display animproved transplantation capacity when implanted in the skeletalmuscle of dystrophin-deficient mdx mice The benefits associatedwith MDSC transplantation are at least partially attributable totheir impressive self-renewal ability and their capacity to undergomultipotent differentiation to form myofibers, blood vessels, andperipheral nerves in the injected muscle We investigated whetherthe injection of normal MDSC clones can promote the regenerationand repair of 3-mm sciatic nerve defects created in mdx mice Wefound that MDSC in the nerve defect area can differentiate intoSchwann cells that significantly promote axonal regeneration andsubsequently alleviate muscle atrophy in denervated gastrocnemiusmuscles The regenerating capacity of the MDSCs in the nervedefect appears to be influenced by the time at which MDSC injection

is performed post-injury Normal MDSC also contribute to theformation of astrocytes and neurons in the hippocampus and cerebralcortex following either intracranial transplantation or intravenousdissemination of the cells These findings demonstrate that MDSCpossess remarkable plasticity in response to environmental cuesand suggest the potential promise of MDSC-based cell therapies fortreatment of various neuromuscular diseases

258 AAV Vector-Mediated Canine Dystrophin Gene Expression in mdx Mice

Mini-Bing Wang,1 Mengnan Tian,1 Chunping Qiao,1 Tong Zhu,1 Juan

Li,1 Xiao Xiao.1,2

1 Dept.of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA, United States; 2 Dept of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, United States.

Duchenne muscular dystrophy (DMD) is the most commondisabling and lethal genetic muscle disorder, affecting one of every3,500 males No effective treatment is currently available for DMD.The mdx mouse has been the most widely used animal model for

DMD, although mdx lacks major clinical deterioration seen in human

patients In contrast, the golden retriever muscular dystrophy(GRMD) dog, as a large animal model, displays remarkable clinicaland pathological similarities to the human DMD patients.Therefore, the GRMD is well conceived as a clinically more relevant

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DMD model Previously, we have attempted AAV-mediated human

mini-dystrophin gene delivery to treat the GRMD, but the result

was not striking We believe that the human dystrophin gene

expression in dog may have elicited immune-response in the

dystrophic dog

In this report, we have cloned the dog dystrophin cDNA via

RT-PCR from normal dog muscle and generated a dog version

mini-dystrophin gene (3.8 kb, including 5 rods) that can be readily package

into AAV vector along with CMV promoter We show that the dog

mini-dystrophin can be expressed in mdx mice at high levels at one

month after vector injection Immunostaining of the consecutive

sections, using antibody against N-terminus of human dystrophin,

revealed the restoration of the missing dystrophin onto the plasma

membrane H & E staining also displayed normal histology and the

lack of fibrosis and infiltration in the vector transduced area The

results prove the feasibility of using AAV dog mini-dystrophin

vector in the large GRMD canine model

259 Lentiviral Vector Mediated Gene Transfer

to Mouse Skeletal Muscle Cells: Potential

Applications for Duchenne Muscular Dystrophy

Sheng Li,1 En Kimura,1 R W Crawford,1 B Fall,1 J M Scott,1 J

C Angello,2 R Welikson,2 S D Hauschka,2 J S Chamberlain.1

1 Department of Neurology, University of Washington School of

Medicine, Seattle, WA; 2 Department of Biochemistry, University of

Washington School of Medicine, Seattle, WA.

Mutations in the dystrophin gene cause Duchenne muscular

dystrophy (DMD) We have shown previously that delivery of

mini- or full-length dystrophin genes to muscles of mdx mice, a

model of DMD, can prevent and partially reverse the dystrophic

pathology However, gene therapy for DMD will require systemic

delivery, and sustained expression, of therapeutic dystrophins in

widely distributed skeletal muscles Lentiviral vectors have a

relatively large transgene carrying capacity and are able to integrate

into non-dividing cells We explored the use of lentiviral vectors for

transferring genes into mouse skeletal muscle cells in vitro and in

vivo The lentiviral vectors efficiently transduced both proliferating

and terminally differentiated mdx muscle cells in vitro, and

transgene-expressing myoblasts were able to differentiate normally without

any obvious toxicity We demonstrated that even a small version of

the murine creatine kinase regulatory cassette maintained

muscle-specific activity in lentivirally-transduced cells We were also able

to transduce with high efficiency the minidystrophin-lentivirus

cassettes into a variety of other cell types in vitro, including

myoblasts derived from dystrophic dogs and hematopoietic stem

cells Although we were able to obtain moderate levels of skeletal

muscle transduction in vivo by direct intramuscular injection, the

relatively low titer of lentiviral preparations combined with physical

barriers to virus diffusion will limit the direct application of lentiviral

vectors for transferring therapeutic genes into muscles Nonetheless,

the ability to successfully transduce both muscle and bone marrow

cells in vitro with mini-dystrophin expressing lentiviral vectors

suggests that this system may have great potential for developing ex

vivo cell therapies for DMD.

260 Functional Analysis of Dystrophin in Vascular Smooth Muscle Cells in Duchenne Muscular Dsytrophy

Kaori Ito,1 Shigemi Kimura,1 Shiro Ozasa,1 Makoto Ikezawa,1

Misao Suzuki,2 Kowashi Yoshioka,1 Makoto Matsukura,1

Takashi Hiranuma,1 Takeshi Miwa,3 Teruhisa Miike.1

1 Department of Child Development, Kumamoto University Medical School, Kumamoto, Kumamoto, Japan; 2 Division of Transgenic Technology Center for Animal Resources and Development (CARD), Kumamoto University, Kumamoto, Kumamoto, Japan; 3 Department of Oncogene Research, Research Insutitute for Microbial Deseases, Osaka University, Suita, Osaka, Japan.

Duchenne muscular dystrophy (DMD) is an X-linked fatal diseasecaused by mutations of the gene encoding the cytoskeletal proteindystrophin Dystrophin is a membrane-associated protein thatprovides a link in a chain of proteins between the actin cytoskeleton

to extracellular matrix and comprises a dystrophin-glycoproteincomplex (DGC) Dispite the abundance of new information on thesemolecules, there currently is no effective treatment for DMD becausethe mechanism by which dystrophin deficiency produces the clinicalphenotype is poorly understood Two principal theories have beenproposed to explain the pathogenesis of DMD The first is thatdystrophin deficiency destabilizes the sarcolemmal integrity, therebyrendering the muscle fibers susceptible to damage duringcontractions The second theory is that disruption of DGC givesrise to the reduction of the scaffolding function that recruits signalingproteins such as neuronal nitric oxyde synthase (nNOS) to themembrane Recent studies indicate that nNOS in skeletal muscleplays a key role in the regulation of the blood flow within exercisingskeletal muscle by blunting the vasoconstrictor response to alpha-adrenergic receptor activation This protective mechanism is defective

in both, DMD patients and mdx mice, an animal model of DMD

We hypothesized that dystrophin deficiency also causes thereduction of nNOS in vascular smooth muscle cells (VSMCs), leads

to vascular dysfunction and exacerbates muscle pathology Wetherefore generated transgenic mice expressing 14Kb full-lengthhuman dystrophin cDNA under the transcriptional control of thesmooth muscle alpha-actin promoter These mice were then crossedwith mdx mice, resulting in three independent SMTg/mdx lines whichharbor the dystrophin gene only in SMCs PCR and southern blotanalysis were performed to verify founders and stable transgeniclines The expression pattern was detectable by semi-quantitativeRT-PCR analysis and immunohistochemical staining, which showedthe specific expression of transgene in SMCs We also report thehistological characteristics of SMTg/mdx mice such as centralnucleation, fiber size variability, and CK concentrations as compared

to C57BL/10 control mice and mdx mice We believe that our SMTg/mdx mouse model is worth exploring to gain a better understanding

of the functon of dystrophin in VSMCs and the pathophysiology

of DMD patients We also believe that the introduction of dystrophingene into VSMCs is necessary for the effective treatment for DMD.References

1) Kobzik L., et al Nature 1994; 8(372):546-48

2) Brenman JE., et al.Cell 1995; 82(5):743-52

3) Chang WJ., et al Proc Natl Acad Sci USA 1996;93(17):9142-47

4) Gail D.Thomas., et al Proc Natl Acad Sci USA 1998;95:15090-95

5) Mikael Sander, et al Proc Natl Acad Sci USA 2000;97(25):13818-23

6) Michelle Wehling, et al J Cell Biol 2001; 155(1):123-31

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261 An AAV Vector-Mediated Micro-Dystrophin

Expression Ameliorates Dystrophic Phenotypes of

mdx Muscles

Miki Sakamoto,1 Madoka Yoshimura,1 Katsutoshi Yuasa,1

Toshifumi Yokota,1 Takaaki Ikemoto,2 Xiao Xiao,3 Yuko

Miyagoe-Suzuki,1 Shin’ichi Takeda.1

1 Molecular Therapy, National Institute of Neuroscience, Kodaira,

Tokyo, Japan; 2 Pharmacology, Saitama Medical School,

Moroyama, Saitama, Japan; 3 Molecular Genetics and

Biochemistry, University of Pittsburgh, Pittsburgh, PA.

Duchenne muscular dystrophy (DMD) is an X-linked, lethal

muscle disorder caused by a defect in the dystrophin gene, and

characterized by progressive muscle weakness, cardiomyopathy

and early death An adeno-associated virus (AAV) vector-mediated

gene transfer is one of attractive approaches for the treatment of

DMD, but it has a limitation in insertion size up to 4.9 kb Therefore,

a full-length dystrophin cDNA (14 kb) cannot be incorporated into

an AAV vector To find a short but functional dystrophin cDNA,

we generated a series of rod-truncated micro-dystrophin (M3, 3.7

kb; AX11, 4.4 kb; CS1, 4.9 kb), and generated transgenic (Tg)

dystrophin-deficient mdx mice expressing each of

micro-dystrophins CS1 Tg mdx mice showed lowest levels of serum

creatine kinase, complete amelioration of muscle pathology, and

nearly full restoration of contractile force (Biochem Biophys Res

Commun 293:1265, 2002) To test whether AAV vector-mediated

CS1 micro-dystrophin expression can ameliorate the dystrophic

phenotypes of mdx muscle, we constructed an AAV vector expressing

micro-dystrophin CS1 We used skeletal muscle-specific MCK

promoter to drive the CS1 gene, since the MCK promoter in AAV

vector drives longer expression of the LacZ gene than the CMV

promoter in skeletal muscle (Gene Ther 23:1576, 2002) To reduce

the length of CS1 cDNA, we deleted 5’- and 3’-untranslated regions

and the coding region corresponding to exons 71- 78 (ΔCS1, 3.8 kb)

injected the AAV vector into anterior tibial (TA) muscles of

10-day-old mdx mice At this age,mdx mice show no signs of muscle

degeneration Therefore, it is easy to evaluate the therapeutic effects

of the vector by counting the ratio of centrally nucleated myofibers

We then injected the vector into 5-week-old mdx mice whose muscles

show active cycles of muscle degeneration/regeneration When the

AAV vector was introduced into 10-day-old mdx mice, the expression

of micro-dystrophin continued for a long time, but

dystrophin-positive fibers scattered; 10 to 23% at 24 weeks after the AAV

injection H&E staining of muscle tissues showed nearly normal

morphology of dystrophin-positive fibers In contrast, extensive

expression of micro-dystrophin was achieved when 5-week-old mdx

muscles were treated At 8 weeks after the AAV vector injection, a

large percentage of fibers were dystrophin-positive (10 to 50%)

Even 24 weeks after the injection, 15 to 75 % of myofibers expressed

micro-dystrophin Dystrophin-positive fibers often had centrally

located nuclei, however, ratio of these fibers was significantly reduced

compared with that of dystrophin-negative fibers We then isolated

measured tetanic force Non-treated mdx muscle showed reduced

specific tetanic force, but AAV-injected mdx TA muscles showed

moderate improvement of the specific force Thus, our study

vector both before the onset of dystrophic changes and during ongoing

muscle degeneration successfully protected mdx muscle.

262 Chronic Inflammation-Induced Extrasynaptic Utrophin Upregulation in Muscle Fibers of Immune Competent mdx Mice Is Related

to Reduced Calpain Activity of Muscle

Ishrat Waheed,1 Renald Gilbert,1 Basil Petrof,2 JosephineNalbantoglu,1 George Karpati.1

1 MNI; 2 RVH.

Chronic inflammation induced in the anterior tibialis muscles ofimmune competent mdx mice by intramuscular injection of a firstgeneration adenovirus (FGAV) with strong beta galactosidaseexpression produces, by 30 days, appreciable amounts ofextrasynaptic utrophin (utr) Utr is close structural and functionalanalogue of dystrophin (dys), and the amount of the extrasynapticutr produced by chronic inflammation is sufficient to mitigate thedeleterious effects of dys deficiency (reduced muscle fiber necrosis,restoration of dys-associated proteins, force generation impairment,etc.) Certain proinflammatory cytokines have been suspected tohave a role in this process which, among other things, is supported

by our finding that in certain types of immune incompetent mice(i.e TNF-alpha gene-ablated), the above-described extrasynapticutr upregulation does not occur By contrast, in the IL-6 gene-ablatedanimals, utr levels remain unaffected, possibly because of vigorouscompensation by IL-6 analogues Here we also report that activity

of calpain was significantly reduced in the inflammatory mdx musclescompared to non-inflammatory mdx controls (p<.0047) We alsodetermined that in myotube cultures, utr is subject to calpain-mediated proteolysis in a calcium-dependent manner Furthermore,certain proinflammatory cytokines (IL-1 beta and IL-6, but notTNF-alpha) inhibited calcium-induced calpain activity

We suggest that inhibition of calcium-dependent muscle calpainactivity by proinflammatory cytokines is related to the increase ofthe extrasynaptic utr, which is normally produced in small quantitiesand is subject to degradation by calpain Inhibition of calpain wouldthus increase the extrasynaptic utr level to a new, higher-than-normal,steady state Thus, inhibition of muscle calpain activity by othermeans may turn out to have a therapeutic role for dys deficiency

263 Transfection of mdx and Normal Murine Muscle Fibers by Electrotransfer of Plasmid Encoding GalNac Transferase

Margaret Durko,1 Renald Gilbert,1 Josephine Nalbantoglu,1

an expression cassette of CMV-murine GNT cDNA (kind gift of

Dr P Martin) and hyaluronidase (30 ul at 0.4 mg/ul) Subsequently,

a train of eight 200 V/cm electrical pulses, each of 20 millisecondduration, was delivered by needle electrodes using an ECMA 30electroporator to the AT muscles Control muscles were not injectedwith plasmid but otherwise treated like the experimental ones Ten-

12 days later, transverse cryostat sections of the treated and control

AT muscles were stained with biotinylated Wisteria Floribunda

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(WFA) or biotynilated Vicia Villosa (VVA) lectins with and without

neuraminidase pretreatment, as well as with an antibody to GNT

(gift of Dr P Martin) and utrophin In several of the treated mdx

and CD1 muscles, large groups of large diameter muscle fibers

showed strong extrajunctional sarcolemmal WFA and VVA binding

Neuraminidase pretreatment increased peripheral staining of

myofibers with WFA and VVA However, the same fibers showed

no extrasynaptic utrophin immunoreactivity even though synaptic

utrophin expression was normal We suggest that lack of

extrasynaptic utrophin expression in the GNT muscle fibers may

be explained by either that, unlike transgenic fibers, GNT-transfected

mature mdx muscle fibers do not express utrophin, or the expression

would take longer than 12 days These points have practical

importance if overexpression of extrasynaptic GNT is contemplated

for the therapy of dystrophin deficiency

264 Gene Transfer of vIL-10 To Improve

Myoblast Survival in MDX Mice

Yong Li,1 William Forster,1 Levent Balkir,2 Paul Robbins,2 Johnny

Huard.1,2

1 Orthopeadic Surgery, University of Pittsburgh, Pittsburgh, PA,

United States; 2 Molecular Genetics and Biochemistry, University

of Pittsburgh, Pittsburgh, PA, United States.

Myoblast transplantation is a potential therapy for Duchenne

muscular dystrophy (DMD) However, the poor survival of

myoblasts following transplantation has hindered the overall

application of this technology Immuno-rejection has been considered

to be a major limitation following myoblast transplantation

Decreased immune rejection is known to increase the number of

myoblasts that survive post-transplantation Viral interleukin-10

(vIL-10) can suppresses autoimmune response after transplantation,

and has already been found to prolong survival rates for different

cells and tissues post transplantation in several animal models We

hypothesis that vIL-10 may improve myoblasts survival after

transplantation due to its immunosuppressive function A plasmid,

encoding vIL-10 gene and the neomycin resistance gene, was

transfected into myoblasts (C2C12) by lipofectin The clone cells

were selected by 500m g/ml G418 for 14 days after gene transfection

ELISA and western blot were performed to ensure that the clone

cells were expressing of vIL-10 Both the selected clone cells

(C2C12vIL-10) and normal myoblasts (C2C12) were retrovirally

transduced to express a LacZ gene and then directly injected into

gastrocnemius muscles (C2C12vIL-10 clone cells in left legs and

C2C12 cells in right legs) of MDX mice (C57BL6J, age 6week) All

mice were sacrificed for histological analysis at different time points

(3days and 1,2,3,5,7 weeks) post cell transplantation We also

performed physiological testing at 7 weeks post transplantation

From counting the LacZ positive myofibers on both sides, we found

that the surviving numbers of C2C12vIL-10 cells were greater than

those of the control C2C12 cells The immunohistochemical results

show that the numbers of dystrophin positive myofibers in the

C2C12vIL-10 injected muscle were significantly high than those of

the control C2C12 injected muscle at different times point post

transplantation This increased survival of myoblasts resulted in

improved MDX mouse muscle strength as assessed by fast twitch

and tetanus strength tests 7 weeks post transplantation Our results

demonstrated that vIL-10 gene transfected myoblasts could survive

longer than control non-gene transfected myoblast in MDX mice

This prolonged survival of transplanted myoblasts subsequently

led to an increase in muscle strength of DMD muscle

Key Words: vIL-10, gene transfer, myoblasts transplantation,

mdx mice

265 The Differentiation of Embryonic Stem Cells into Muscle Cells with Tet-Off System for Duchenne Muscular Dystrophy Therapy

Shiro Ozasa,1 Shigemi Kimura,1 Kaoru Ito,1 Makoto Ikezasa,1

Takashi Hiranuma,1 Makoto Matsukura,1 Kowashi Yoshioka,1

Teruhisa Miike,1 Kimi Araki,2 Kuniya Abe,2 Kenichi Yamamura,2

Hitoshi Niwa.3

1 Child Development, Kumamoto University School of Medicine, Kumamoto, Kumamoto, Japan; 2 Department of Developmental Genetics, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Kumamoto, Japan;

3 Laboratory for Pluripotent Cell Studies, RIKEN Center for Developmental Biology, Kobe, Hyogo, Japan.

Introduction

Duchenne muscular dystrophy (DMD) is an X-linked recessivemuscle disease caused by the deficiency of dystrophin Variousapproaches, including cell transplantation and gene therapy, havebeen carried out in order to restore the missing dystrophin gene inDMD patients Although gene therapy for DMD has shown somepromise, viral vectors are limited to the injected area and we believethat DMD is a systemic disease Conversely, muscle and/or bonemarrow derived stem cell transplantation to dystrophic muscle bysystemic, intravenous injections has succeeded for the delivery ofdystrophin (1, 2) These cells cannot retain their stem cellcharacteristics if cultured for a long time In contrast, embryonicstem (ES) cells can be cultured for a long time, keeping their originalcondition and having the capacity to differentiate into all tissues andcell types ES cells induced to differentiate in vitro gave rise to manycell types including hematopoietic precursors, cardiac and skeletalmuscle, endothelium, and neural cells Previously, it was impossible

to limit differentiation of the ES cells to the muscle lineage We haveshown that ES cells can be induced to differentiate into myotubes

by the establishment of genetically engineered ES cells harboring atetracycline-regulated expression (Tet-Off System)(4) vectorencoding the myogenic transcriptional factor, MyoD (3)

Methods

Supertargeting is a good strategy to make Tet-Off System-regulated

ES cells ZHTc6 is a mouse-derived feeder-free ES cell line, whichcarries Oct-3/4 cDNA regulated by the Tet-Off System In the Tet-Off System, a cDNA artificially drives expression by the removal

of tetracycline The supertargeting vector, which was designed toreplace the Oct-3/4 with MyoD cDNA and have neomycin resistantgene expression, was constructed for the supertargeting.Electroporation of the supertargeting vector to ZHTc6 and subsequentG418 selection were performed Subsequently, 6 colonies wereisolated, picked up, and screened by Southern blot analysis

Results

The Southern blot analysis showed that the Oct-3/4 cDNA in 3clones was replaced by MyoD cDNA by homologous recombination.The clones were then induced to differentiate into myotubes followingthe removal of tetracycline

Discussion

In this system, we can maintain ES cells undifferentiated in thepresence of tetracycline and induce them to differentiate exclusivelyinto myogenic pathways by the removal of tetracycline in vitro.Further in vivo and in vitro analysis is needed to delineate thepossibilities of the clinical application of this system

References

1.Gussoni E et al Nature 1999 Sep 23;401(6751):390-4 2.Ferrari G et al Science 1998 Mar 6;279(5356):1528-30 3.Pinney DF et al Cell 1988 Jun 3;53(5):781-93 4.Niwa H et al Nat Genet 2000 Apr;24(4):372-6

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266 Tolerance Induction by a Plasmid

Expressing Anti-CTLA4-Antibody

An-Bang Liu,1 Steve R Roffler.2

1 Neurology, Tzu-Chi Medical Center, Hualien, Taiwan; 2 Institute

of Biomedical Science, Academia Sinica, Taipei, Taiwan.

Background and Aims: Host immune responses against either

the vector gene products or transgenes themselves are the major

causes to decrease long-term expression of the transgenes in gene

therapy study To attenuate the host immune responses, we

co-administrated a plasmid expressing a stably membrane anchored

single-chain antibody, anti-CTLA4-mAb, with another plasmid

expressing E coli beta-galactosidase gene The latter was used to

trigger host immune responses against the transduced cells Methods:

Two groups of 9-week old adult female C57BL/6 mice were used in

this study The first group, 10 mice, received intramuscular injection

phosphate buffer) on the left anterior tibialis The other group

received co-administration of a plasmid encoding anti-CTLA4-mAb

with the same volume and same concentration on the left anterior

tibialis Results: Half of the mice were sacrificed at 15th day of

injection and the rest were killed at 30th day of study Expression of

beta-galactosidase, host cellular and humoral immunities were

evaluated on cryosection of the injected muscles The average number

of X-gal stained muscle fibers in each cryosection is 8.7 in the

co-injected mice, which is higher than that, 0.48, in the little mates

Neutralization antibodies against beta-galactosidase in the host serum

were detected by goat antimouse IgG conjugated with horseradish

peroxidase The absorbance at 406 nm is 2.23 in the control group as

compared with 1.54 in the mice receiving two plasmids The p value

is 0.019 by paired Student’s τ-test Infiltration of CD4+ and CD 8+

cells are less prominent in the mice co-injected with

anti-CTLA4-mAb However there is no statistic significance between these two

groups Conclusions: In this study, we demonstrate that

co-administration of anti-CTLA4-mAv can prolong the expression of a

foreign gene in immune-competent animals Surface expression of

this single-chain antibody against CTLA4 can upregulate the function

of the inducible T-cell receptor, CD 152, and then inhibit activated

T-cells Co-administrating or inserting the anti-CTLA4-mAb into a

dicistronic expression cassette may increase long-term expression

of the transgenes It can be very useful in gene therapy of the diseases

with genetic defects or promote the application of viral vectors,

such as the first-generation adenoviral vectors

267 Matrylisin over Expression Enhance the In

Vivo Migration of Myoblasts

Jean-Francois Lafreniere,1 Philippe Mills,1 E Mostafa El

Fahime,1 Jacques P Tremblay.1

1 Genetique Humaine, Centre de Recherche du CHUL, Ste-Foy,

QC, Canada.

The success of myoblast transplantation as a potential treatment

for Duchenne muscular dystrophy (DMD) has been limited in part

by the low dispersion of transplanted myoblasts Injection sites at

every millimeters is required to provide a good transplantation

success We previously showed that in vivo myoblast migration

throughout the muscle extracellular matrix requires matrix

metalloproteinase activity We now demonstrate that over-expression

of matrilysin (MMP7) could helps to reduce the number of injection

sites for the treatment of DMD patients

We constructed a retroviral vector including the matrilysin cDNA

RT-PCR and Western blots were used to verify that the retrovirus is

effective and that the protein is expressed and secreted by the

infected cells After the evaluation of the infection rate by

immunocytochemistry, the infected mouse myoblasts were

transplanted and the in vivo migration was quantified after 60 hours

by the micro-tube technique [ El Fahime E et al : Exp Cell Res,

2000 ]Our results show that the over-expression of MMP7 improvesthe in vivo migration of mouse myoblasts After 60 hours, themigration distance of normal mouse myoblasts is about 340 μm.After infection of only 25% of the cells with the retrovirus, themigration distance reach 961μm

These results suggest that matrilysin over-expression is a effectiveway to improve intramuscular migration and reduce the number ofinjection sites required for a successful myoblast transplantation

268 Muscle Stem Cells Can Be Used as Antigen Presenting Cells in Ex Vivo Gene Transfer

to Skeletal Muscle Mediated by Adenovirus

Baohong Cao,1 Ryan Pruchnic,1 Mike Epperly,2 Thomas J.Wickham,3 Imre Kovesdi,3 Zhuqing Qu-Petersen,1 JohnnyHuard.1

1 Orthopaedic Surgery, Children’s Hospital of Pittsburgh, Pittsburgh, PA, United States; 2 Radiology, University of Pittsburgh, Pittsburgh, PA; 3 GenVec, Inc, Gaithersburgh, MD.

The virus’ inability to transduce mature muscle fibers as well asthe immune response represent two major limitations facing theoverall application of adenoviral (AD) mediated gene transfer toskeletal muscle It has been recently shown that a reduction of theimmune response as well as an improvement of gene transfer inmature muscle fibers can be achieved through the use of a muscle-specific promoter that restricts the transgene expression specifically

to muscle fibers We have therefore investigated whether the sameeffect can be achieved through ex vivo gene transfer using musclederived stem cells (MDSC) Two adenoviral vectors: adenovirusencoding luciferase gene under cytomegalovirus (CMV) (ADCMV)

or under muscle creatine kinase (MCK) promoters (ADMCK) wereused We showed that the ex vivo gene transfer through adenovirally(ADCMV) transduced MDSC triggered a higher immune response

as well as a shorter transgene expression when compared to theADMCK transduced MDSC We have observed that the ADCMV-transduced MDSC cells in the ex vivo gene transfer approach canacts as antigen presenting cells (APCs) by expressing the transgeneand therefore rapidly initiate the immune response In contrast, theADMCK transduced MDSC express the transgene only upondifferentiation into myotubes and myofibers that lead to a longerpersistence of the transgene in the injected skeletal muscle Theseresults demonstrate that the use of a muscle specific promoter thatrestrict the transgene expression specifically in muscle fibers isimportant to prevent the MDSC to become APC following ex vivogene transfer to skeletal muscle

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269 Inactivating Mutation in the MYH 16

“Superfast” Myosin Gene Abruptly Reduced the

Size of the Jaw Closing Muscles in a Recent

Human Ancestor

Hansell H Stedman,1,3 Benjamin W Kozyak,1 Anthony Nelson,1

Danielle M Thesier,4 Joseph B Shrager,1,3 Charles R Bridges,1

Nancy Minugh-Purvis,2 Marilyn A Mitchell.1

1 Department of Surgery, University of Pennsylvania School of

Medicine, Philadelphia, PA, United States; 2 Department of

Developmental and Cell Biology, University of Pennsylvania

School of Medicine, Philadelphia, PA, United States;

3 Pennsylvania Muscle Institute, University of Pennsylvania School

of Medicine, Philadelphia, PA, United States; 4 Department of

Cellular and Molecular Biology, University of Pennsylvania,

Philadelphia, PA, United States.

The hominid fossil record reveals dramatic differences in the size

of the masticatory muscles in most members of the genus Homo

compared with extinct known genera of the family Hominidae

Various species of Australopithecus and Paranthropus exhibit, among

other features, temporal fossae volumetrically resembling those of

Pan troglodytes and Gorilla gorilla and considerably exceeding those

of fossils attributed to early Homo and living humans alike We

show that the reduction in overall masticatory muscle size can be

attributed to dramatic trophic changes in a distinctive class of muscle

fibers expressing transcripts of the recently discovered MYH 16

gene In the diminutive type II fibers of the modern human temporalis

muscle, translation of the MYH 16 transcript is blocked by a

frameshifting mutation This micro-deletion is unique among modern

primates to Homo sapiens Using the coding sequences for myosin

rod domains as a molecular yardstick, we estimate the appearance

of this mutation at 2.5 mya, immediately predating the appearance

in the paleoanthropological record of modern human body size and

the emigration from Africa of an early species of Homo, possibly

Homo ergaster This represents the first proteomic distinction

between Homo and the great apes that can be correlated with a

traceable anatomic imprint in the fossil record

270 Skeletal Tissue Engineering Applications

of Multipotential Mesenchymal Stem Cells Derived from Adult Human Tissues

of skeletal tissues, many of these applications include the use ofthree-dimensional biomaterial scaffolds that have physical andchemical properties optimal for the specific tissue(s) being engineered.Some of these properties include porosity, biodegradability,conjugation with bioactive ligands, and biocompatibility for celladhesion and proliferation Another requirement for successful tissueengineering using MSC is the use of bioactive and signaling moleculesthat optimally regulate cellular differentiation into the appropriatephenotype Introduction of these agents may be accomplished bythe direct incorporation of recombinant growth factors or signalingmolecules into the tissue construct, or by means of geneticmanipulation of the tissue progenitor cells, such as the MSC Theefficiency of MSC transfection is generally recognized to beinefficient, and currently, adenoviral and retroviral vectors are mostfrequently used for gene transduction of MSC For skeletal tissueengineering, the target is most commonly the expression of growthfactors and signaling molecules, such as members of the bonemorphogenetic protein (BMP) family, vasculoendothelial growthfactor (VEGF), insulin-like growth factors (IGF), and others Whilethe majority of procedures in tissue engineering being tested are exvivo manipulations using exogenous cells, there are also otherattempts directed towards in situ gene transduction of local tissueprogenitor cells, using microprojectile-mediated methods or gene-activated matrices Successful MSC-based tissue engineering is thuscontingent on a number of factors: 1) elucidation of mesenchymaldifferentiation pathways and identification of key regulatory genes;2) rapid and efficient isolation and culture expansion of MSC; 3)design of bioactive and biodegradable biomaterial scaffold for tissue-specific applications; and 4) efficient gene transduction for theregulated expression of tissue-specific growth factors and signalingmolecules The overwhelming burden of disease associated withmusculoskeletal disorders underscores the urgency of MSC-basedskeletal tissue engineering

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271 Fetal Gene Transfer Using Lentiviral

Vectors for Heart, Lung, and Blood Diseases:

Assessments of Safety and Efficiency in Rhesus

Monkeys

Alice F Tarantal,1 Daniel F Jimenez,1 Chang I Lee,1 Ruth J

McDonald,1 Kristen R Kralovich,1 John J Rossi,2 Donald B

Kohn.3

1 California National Primate Research Center, University of

California, Davis, CA; 2 Molecular Biology, Beckman Institute of

the City of Hope, Duarte, CA; 3 Research Immunology/BMT,

Childrens Hospital Los Angeles, Los Angeles, CA.

We are exploring fetal gene transfer in rhesus monkeys for heart,

lung, and blood applications, with a focus on the safety of these

techniques for the fetus, infant, and dam Viral vectors under

investigation include SIN VSV-G pseudotyped HIV-1-derived

lentivirus using EGFP as a reporter gene For heart and lung

applications, fetuses were transferred at 60 or 70 days gestation

(early 2nd trimester; term 165±10 days), respectively, using a direct

intraorgan administration approach (107 infectious particles/fetus)

For blood-related diseases, intraperitoneal administration at 50 days

gestation or intra-yolk sac injection at 20 days gestation were also

explored All fetuses (N=15) were monitored sonographically and

delivered by cesarean-section, then newborns nursery-reared for

postnatal studies All animals remained healthy during the study

period (growth, development, hematology, clinical chemistry), with

no evidence of adverse effects Prenatal and postnatal

echocardiographic profiles for animals that were transferred using

an intramyocardial approach were within normal limits when

compared to controls Whole tissue fluorescence microscopy on the

day of harvest showed strong transgene expression within the

myocardium and pericardium, with high copy numbers when assessed

using a quantitative PCR assay (1.5x106 copies/104 cells; 10%)

EGFP was also detected in lung lobes, thoracic wall, and diaphragm

(1-18%) For the animals transferred using an intrapulmonary

approach, pulmonary function tests were performed at 1 mo of age

(4 mos post-intrapulmonary gene transfer) and found to be within

normal limits when compared to controls Fluorescence microscopy

of whole tissue on the day of harvest showed strong transgene

expression for all animals Collected tissues were analyzed using

quantitative PCR and results showed high copy numbers for all lung

lobes (34-909 copies/104 cells; 10-35%) with evidence of EGFP in

postnatal animals at the time of tissue harvest (6 mos post-gene

transfer) indicated no differences when compared to controls The

results of these studies are significant because they indicate that

postnatal heart and lung development and function were not altered

after direct intraorgan gene delivery For blood-related applications,

one group was transferred with a VSV-G pseudotyped

HIV-1-derived lentiviral vector (HIV-7) expressing the TAR decoy and the

ribozyme against the CCR5 co-receptor

(pHIV-U6U16TAR-VACCR5-EGFP) Results post-transfer indicated gene expression

by flow cytometry (~10%) and fetal hematopoietic progenitors

with high levels of transduction (~50%) Analysis of immunoselected

cells from cord blood (CD2, CD4, CD8, CD19, CD34) and marrow

(CD34) at delivery revealed high copy numbers in all cell types

using a quantitative PCR assay (1x10²-1.8x10³ copies/104 cells)

These animals are currently 6 mos postnatal age and are healthy

with no evidence of adverse effects Studies with the monkey are

essential for identifying efficient gene transfer strategies, and are

crucial for determining whether such treatment approaches will be

safe for use in humans

272 A Clinically Applicable Protocol for Producing Oligonucleotide Directed Single Base Pair Changes in Human Hematopoietic Stem/ Progenitor Cells

Wei Han,1 Barbara Fish,1 Matthew Chomo,1 Margaret Wong,1

a T (sickle mutation) in codon 6 of beta-globin are electroporated

Quantitation of fluorescent conjugated-oligonucleotide uptake andkinetics in the CD34+ cells are determined by fluorescent activatedcell sorting (FACS) The efficiency of conversion of the normal A tothe mutant T in CD34+ cells is screened by Snapshot/sequencing of

a PCR amplified 352 bp beta-globin fragment flanking codon 6 Theproduction of sickle hemoglobin in cytokine stimulated red bloodcells is quantitated with a fluorescent conjugated sickle specificmonoclonal antibody Results: (1) We achieved up to 80% oligouptake in CD34+ cells We defined optimal voltage and durationconditions for electroporation of oligonucleotides into cells The

oligonucleotide uptake significantly (2) We achieved 3.6% ± 3.2%(Mean ± SD, N = 5) targeted alteration in CD34+ cells We alsodiscovered that multiple factors affected the efficiency of the genecorrection including oligo length, chemical modification,oligonucleotide purity and quality, as well as the length of cultureand cytokine stimulation of CD34+ cells In our preliminary studies,

we detected the change of A to T in a colony-forming-unit

cells 9 weeks post-electroporation (3) Red blood cells are obtained

(Kit ligand), Flt-3 ligand, Thrombopoietin, and Erythropoietin Inour preliminary studies, we detected sickle hemoglobin (HbS)production in the red blood cells using a monoclonal anti-HbSantibody Conclusion: We have developed a clinically applicableprotocol using synthetic oligonucleotides to achieve allele-specificgene alteration in human HS/PCs

273 Highly Efficient Gene Transfer to Dog Repopulating Cells Using Concentrated RD114 Pseudotype Oncoretroviral Vectors

Tobias Neff,1 Peter A Horn,2 Jesse Thompson,1 Laura J.Peterson,1 Julia C Morris,1 Hans-Peter Kiem.1

1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 2 Institute for Transplantation Diagnostics and Cell Therapeutics, Institute for Transplantation Diagnostics and Cell Therapeutics, Dusseldorf.

The receptor for the feline endogenous retrovirus RD114 hasbeen shown to be a promising molecular target for oncoretroviralvectors RD114 pseudotype vectors have been shown to efficientlytransduce canine hematopoietic repopulating cells and human NOD/

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SCID-repopulating cells A particularly attractive feature of RD114

pseudotype vectors is the possibility to concentrate the viral particles

by centrifugation This has two important advantages Firstly, higher

vector titers achieved by centrifugation and resuspension in a smaller

volume could potentially lead to higher gene transfer levels, saturating

the receptors expressed on stem cells Secondly, the concentration

and resuspension may significantly deplete unwanted as well as

toxic by-products present in the virally conditioned medium

harvested from producer cell lines Because conditioned medium

from the original RD114 producer cell line, FlyRD, has been shown

to exert differentiating activity on repopulating hematopoietic cells,

we have generated a novel RD114 pseudotype producer cell line

based on 293T cells, Phoenix-RD114

Preliminary in vitro studies suggest that gene transfer level to

canine CD34-selected cells is higher (around 90%) for unconcentrated

Phoenix-RD114 derived vector compared to FlyRD derived vector

(around 80%), despite a 10-fold higher titer of the FlyRD producer

clone To evaluate Phoenix-RD114 derived vector in vivo, we

transplanted three dogs with autologous CD34 selected cells

transduced with concentrated RD114-pseudotype vector In the

first two dogs, cGCSF/cSCF-primed bone marrow was used as source

of stem cells In the third dog, cGCSF/cSCF-mobilized PBSC were

used CD34+ cell dose was between 6.4 and 7.8 million /kg

bodyweight All three dogs engrafted (ANC > 500/μl between d8

and d29) and showed a peak marking in granulocytes of 35% - 49%

early after transplantation Marking stabilized in the first two dogs

after 95-105 days with levels of 16 and 17% in granulocytes, 5%

and 10% in red blood cells and 5% and 10% in platelets In the third

animal, with a shorter follow up, marking at 60 days post

transplantation is 10% in granulocytes Multilineage marking was

confirmed by using antibodies to lineage specific antigens CD3,

CD13, CD14 and CD21 The second dog succumbed to pancreatitis

on day 144 post transplantation The other two animals are alive

and well These data compare favorably with our previous data

evaluating FlyRD based vector in the canine system where results

were hampered by inconsistent engraftment and highly variable in

vivo marking Phoenix-RD114 derived vectors also efficiently

transduce human CD34-selected cells (gene transfer rates of

70%-95% as assessed by flow cytometry on day 10 post transduction)

Therefore, concentrated viral vector preparations hold great promise

for safer and more efficient in vivo gene transfer In summary, we

show that concentrated RD114-pseudotype vectors produced by

human Phoenix cells allow for highly efficient, consistent gene

transfer in a canine in vivo model of stem cell gene therapy and in

human CD34-selected cells in vitro

274 Lentiviral Gene Transfer into

Hematopoietic Stem Cells: Getting the Best out of

It

Gustavo Mostoslavsky,1 John T Gray,2 Richard C Mulligan.1

1 Medicine, Children’s Hospital, Boston, MA; 2 Harvard Gene

Therapy Initiative, Harvard Institute of Medicine, Boston, MA.

Genetic modification of hematopoietic stem cells (HSC) is an

appealing approach for the correction of genetic diseases of the

hematopoietic system To this end, the relevant gene must be stably

expressed in cells that are capable of both self-renewal and

differentiation into all hematopoietic lineages In the past we have

identified a highly enriched HSC population, termed SP, that is

capable of reconstituting myeloablated mice One of the interests of

our lab is to study the introduction of different gene products into

HSC in order to manipulate their fate by altering their genetic material

For this purpose it is imperative that all in vitro procedures minimize

their impact on the engraftment potential of HSC In recent years,

several groups have used retroviral and lentiviral vectors to effectively

transduce HSC however, the transduction protocols used may have

affected the engraftment potential and viability of the transducedstem cells In addition, the use of highly heterogeneous stem cellpopulations made transduction efficiency difficult to assess.The aim of our study is to optimize a transduction protocol inwhich the engraftment capacity of transduced cells is identical tounmanipulated cells and the reconstituting cells permanently expressthe delivered gene Although in vitro assays exist to identifyprogenitor cells, they do not correlate well with true HSC potential

in vivo For this reason, we have used SP cells in competitiverepopulation assays to reconstitute myeloablated mice, and we havebeen able to follow in detail long-term HSC activity

Purified SP cells were transduced either for four hours or overnight,

on ice or at 37°C, using serum free media conditioned with SCF andTPO First we used a lentivirus encoding GFP to mark the transducedcells Optimal transduction was achieved during overnight incubation

at 37°C This transduction protocol did not alter the engraftmentpotential of donor cells compared to mock infected or unmanipulatedcells When 200 SP cells were transduced and injected in competitionwith 2x105 whole marrow competitor cells, the majority of cells inthe peripheral blood of the transplanted animals were of donororigin More importantly, between 75% to 90% of these cellsexpressed the delivered gene for the duration of this study (24 weekspost bone marrow transplantation) Moreover, all blood lineages(e.g B220, CD3, Mac-1, Gr-1 and TER-119 cells) expressed GFPdemonstrating that the real long-term HSC were efficientlytransduced We then compared the use of retroviruses to lentiviruses

promoters, and studied their capacity to transduce HSC In all cases,lentiviral vectors, but not retroviral vectors, efficiently transducedHSC Although all promoters were able to drive GFP expression inperipheral blood cells, the CMV promoter showed the strongestactivity Finally, by using escalating MOIs, we demonstrated thathigh MOI dramatically decreased the engraftment potential of HSC

In summary, we describe an optimized protocol to efficientlytransduce long-term HSC without affecting their engraftmentpotential, and by using this protocol we demonstrate that lentiviralvectors rather than retroviral vectors can be used as a potent tool forgene transfer into HSC

275 Comparison with c-Mpl and c-Kit Mutants and Identification of a Genetic Modifier Locus That Modulates the Severity of Hematopoietic Stem Cell Dysfunction in Mice Lacking Expression

of STAT5

Heath L Bradley,1 Christine Couldrey,1 Kevin D Bunting.1,2

1 Hematopoiesis Department, American Red Cross Biomedical Research and Development, Rockville, MD; 2 Anatomy and Cell Biology, The George Washington University, Washington, DC.

Cytokine stimulation promotes efficient retroviral-mediated genetransfer into hematopoietic stem cells (HSCs), but the intracellularsignals downstream of cytokine receptors are not well characterized

We have demonstrated previously that C57Bl/6 mice lackingexpression of signal transducer and activator of transcription-5(STAT5) display severe defects in competitive HSC repopulatingfunction However, in the absence of a competitor, bone marrow(BM) cells from mice lacking both STAT5a and STAT5b could fullyreconstitute all but T cell lineages, in primary and secondary irradiatedrecipients Here we tested stress hematopoiesis by serial transplant

of STAT5-deficient BM cells in the absence of a competitor andobserved a dramatic loss of contribution to tertiary recipients(0.8 ± 0.5% for STAT5ab-/- vs 82 ± 7% for +/+), despite high levelengraftment of all secondary recipients In order to determine howdefects in STAT5-deficient HSCs compared with other stem celldefective mice, we have directly and indirectly compared HSC

Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts S109

thrombopoietin or stem cell factor receptor signaling respectively

When mixed 5:1 with wild-type (+/+) CD45.1 competitor BM cells,

wild-type BM cells gave 69 ± 11%, c-Mpl-/- BM gave 33 ± 6%, and

blood CD45.2+ cells with similar results obtained in Gr-1+, B220+,

directly with c-kit signaling defective mice, non-irradiated W/Wv

mice were injected with 20-25% BM volumes of STAT5ab-/-or

wild-type cells All primary recipient mice were fully engrafted with

donor cells in mature red blood cells (hemoglobin electrophoresis)

and in BM and spleen tissues (Southern blot) Interestingly,

engraftment, whereas wild-type BM fully reconstituted secondary

recipients To further characterize STAT5 function, we also generated

a congenic STAT5ab-/- mouse on the B6.C-TyrcH1bHbbd/By (HW80)

background which contains an ~16 Mb Balb/c-derived insertion from

the albino (Tyr) to the hemoglobin (Hbb) locus of chromosome 7

Interestingly, BM cells from these mice could only partially

reconstitute long-term multilineage hematopoiesis in red blood cells

and B220+, CD4+, Ter119+, and Gr-1+ peripheral blood leukocytes

of irradiated recipients Also, BM cells from these mice showed

<10% red blood cell reconstitution in primary W/Wv recipients and

were non-competitive when competed 1:1 against c-Mpl-/- BM cells

into irradiated recipients The greater HSC repopulating deficiency

on the HW80 congenic background indicates that a modifier gene

present in a defined region of chromosome 7 may interact with

STAT5 signaling pathways in HSCs Furthermore, the greater

hosts supports a model whereby activation of STAT5 in multiple

synergistic pathways promotes HSC self-renewal and competitive

repopulating ability STAT5 may thus represent a central target for

strategies to optimize HSC gene therapy

276 CXCR4-Transgene Over-Expression in

Peripheral Blood CD34+ Cells Enhances Migration

towards SDF-1 α αα αα and Preserves Engraftment

Potential of Cultured NOD/SCID Repopulating

Cells

Sebastian Brenner,1 Narda Whiting-Theobald,1 Joan Sechler,1

Philip M Murphy,1 Andrew G Rudikoff,1 Harry L Malech.1

1 Laboratory of Host Defenses, National Institute of Allergy and

Infectious Diseases, NIH, Bethesda, MD, United States.

The in vivo outcome of ex vivo hematopoietic stem cell gene

therapy depends not only on transduction efficiency, but also on

the efficient engraftment of the transgene expressing stem cells It

has been shown that hematopoietic stem cells progressively lose

marrow reconstitution potential during the ex vivo culture period

that is required for onco-retrovirus integration and that is helpful

even to lentivirus integration This loss of reconstitution potential

may be due to differentiation and/or loss of homing/engraftment

potential Previous studies have shown that primitive CD34+ cells

express the chemokine receptor CXCR4, and there is a correlation

between levels of native expression of this receptor and migration

1999;283;845-8) To investigate whether constitutive over-expression of

CXCR4-transgene in ex vivo cultured mobilized peripheral blood

hematopoietic CD34+ stem cells (CD34+PBSC) results in enhanced

constructed an MFGS-CXCR4 onco-retroviral vector pseudotyped

and 3 after initiation of ex vivo culture with RD114-MFGS-CXCR4

over-expressed the transgene in 〉 90% of cells on day 6, where the

mean fluorescence intensity of the transduced population was 9fold greater than native expression The CXCR4-transduced

170% of naive cultured CD34+PBSC in a fura-2 fluorescence assay

in response to SDF-1α (ATP response was equal for the 2 groups)

In a SDF-1α induced transwell migration assay the CXCR4-transgene

(N=3) than naive cells or cells transduced with another transgene(gp91phox) The calcium flux and migration assays demonstratedfunctionality of transgenic CXCR4 To investigate whetherengraftment could be enhanced by CXCR4-transgene expression of

overnight x4 (days 2-6) with the RD114-MFGS-CXCR4 vector

vector (92% gp91phox expression) The 6 day culture period was atime during which there is a significant decrease in native CXCR4expression and significant impairment of NOD/SCID engraftment

by cultured non-transduced human CD34+PBSC On the 6th day ofculture each NOD/SCID mouse was injected with cells derived from1x106 CD34+PBSC at culture initiation Nine weeks after transfusion

of CXCR4 or control (gp91phox)-transduced CD34+PBSC cells onlyNOD/SCID mice transplanted with CXCR4-transgene expressingcells demonstrated significant human cell engraftment [0.65 +/- 0.1

% human cell engraftment, (N=3)] We conclude that transductionmediated over expression of CXCR4 can preserve the homing/engraftment potential of CD34+PBSC that would normally lose thispotential during prolonged ex vivo culture

277 Non-Invasive Imaging of the Transplanted Bone Marrow Progenitor Cells Transfected with a Triple Gene Reporter System in Fusion with Metotrexate Resistant DHFR In Vivo

Budak-Alpdogan,4 Vladimir Ponomarev,1 Ronald Blasberg,2 DebabrataBanerjee,4 Juri Gelovani (Tjuvajev).1

1 Radiology, Memorial Sloan Kettering Cencer Center, New York, NY; 2 Neurology, Memorial Sloan Kettering Cencer Center, New York, NY; 3 The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey, New Brunswick, NJ;

4 Human Genetics, Memorial Sloan Kettering Cencer Center, New York, NY.

Objectives: Ex vivo transfection of the harvested bone marrow

progenitor cells (BMPC) with a mutant methotrexate resistant DHFRwas shown to enhance therapeutic options for posttransplant tumorrelapses Simultaneous introduction of multiple reporter genes allowsfor non-invasive monitoring of BMPC migration, engraftment andreconstitution of hematopoiesis, using radioscintigraphy, fluorescenceand bioluminescent imaging (BLI)

Methods: Bone marrow, harvested from C57BL/6-Ly.1 donor

mice, was negatively sorted for differentiation determined cells Theenriched population of precursor cells was stably transfected withthe ecotropic retrovirus carrying dmDHFR-TK-GFP-Luc

quadruple fusion gene Transfected cells were positively selected

by FACS using GFP-expression signal and assessed in vitro for thelevel of expression of luciferase and HSV-TK reporter genes 104

BMPC were infused to rescue lethally irradiated C57BL/6-Ly.2

recipient mice D-luciferin was either administered in vivo, immediately post-transplant, or added to BMPC ex vivo, prior to

injection BLI was started in 5 minutes after BMPC injection Forthe follow-up studies, D-luciferin 150 mg/kg was administered I.V

to all animals Control group included irradiated animals withoutBMPC support

Results: Early BLI revealed a weak, but distinctive signal in the

projection of lungs and mediastinum in supine position The higher

initial signal was registered in the animals injected with the ex vivo

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S110

D-luciferin preincubated BMPC Dilution of BMPC local

concentration resulted in disappearance of signal between 36-72

hours Later observations demonstrated BMPC localization to the

final hematopoietic sites: spleen, sternum, femur, tibia and spine

No signal was identified in the control group Weak sources of

photons associated with the projection of open skin/mucosa areas

corresponded to the subinfrared thermal photon background PET

imaging with [18F]FIAU is in progress

Conclusions: Migration of BMPC can be efficiently monitored

in vivo using highly sensitive BLI allowing the imaging of a small

number of cells Lower limits for BMPC dose for BLI were

identified Triple reporter-gene system reduced ex vivo BMPC

culture maintenance time and provided several non-invasive imaging

options including BLI, fluorescence and PET

278 A Clinical Study of MDR1 Gene Therapy

Against Breast Cancer Showed the In Vivo

Expansion of the MDR1-Transduced Normal

Hematopoietic Cells by Docetaxel

Yoshikazu Sugimoto,1 Shunji Takahashi,2 Junko Mitsuhashi,2

Minoru Nakane,1 Satomi Tsukahara,1 Tetsuko Nagamine,2 Sayuri

Minowa,2 Harumi Shibata,2 Yoshinori Ito,2 Keisuke Aiba,2

Kiyohiko Hatake,2 Takashi Tsuruo.3,4

1 Division of Molecular Biotherapy, Cancer Chemotherapy Center,

Jpn Fdn Cancer Res., Tokyo, Japan; 2 Division of Clinical

Chemotherapy, Cancer Chemotherapy Center, Jpn Fdn Cancer

Res., Tokyo, Japan; 3 Division of Experimental Chemotherapy,

Cancer Chemotherapy Center, Jpn Fdn Cancer Res., Tokyo,

Japan; 4 Institute of Molecular and Cellular Biosciences,

University of Tokyo, Tokyo, Japan.

The MDR1 gene encodes the plasma membrane P-glycoprotein

(P-gp) that acts as an ATP-dependent efflux pump for various

antitumor agents Transduction of the hematopoietic progenitor/

stem cells of cancer patients with the MDR1 gene would be an

attractive strategy to treat the patients with anticancer agents without

life-threatening myelosuppression Here we show some promising

results from our clinical study

Two recurrent breast cancer patients were enrolled in this study

After obtaining informed consent, peripheral blood stem cells were

harvested from the patients and about one third of those cells were

enriched for CD34+ cells The CD34+ cells were transduced with

HaMDR retrovirus supernatant in the presence of 5 cytokines (SCF,

FL-ligand, IL-6, sIL-6R, TPO) and fibronectin fragment CH-296

(Takara Bio, Otsu, Japan) Transduction efficiencies ex vivo were

13-17 % in FACS analysis using MRK16 antibody

In April 2001, the 1st patient received high dose chemotherapy

and the transplantation of the MDR1-transduced CD34+ cells and

unmodified peripheral blood stem cells The transplanted

P-gp-positive cells were 22 millions, which were 7 % of whole transplanted

CD34+ cells One week after the transplantation, 3-4 % of the

peripheral blood mononuclear cells were P-gp-positive The ratio of

P-gp-positive cells decreased to 1 % in 2 months Then the patient

received 10 cycles of consolidation chemotherapy with docetaxel

(DTX) every 3-6 weeks (1) In the 1st or 2nd cycles of DTX

treatment, the ratios of P-gp-positive cells in the peripheral blood

prior to the chemotherapy were 1 % They increased to 2-3 % right

after DTX treatment These increases were transient, and the

P-gp-positive cells decreased to the previous levels within 2 weeks (2) In

the 4th to 10th cycles of DTX treatment, the ratios of P-gp-positive

cells prior to the chemotherapy were 2-4 % They increased to 5-10

% after DTX treatment, but they decreased to 2-4 % within 2

weeks (3) Ten months after the 10th DTX treatment, 1-3 % of the

peripheral white blood cells were still P-gp-positive These results

suggest that MDR1-transduced cells were selectively enriched in

vivo by DTX treatment.

The 2nd patient received the transplantation of the

MDR1-transduced cells in October 2001 One week after the transplantation,3.3 % of the peripheral blood mononuclear cells were P-gp-positive.This patient was not treated with DTX for 7 months The P-gp-positive cells decreased, and almost disappeared before the start ofDTX treatment Then the patient received 5 cycles of consolidationchemotherapy with DTX After the DTX treatments, P-gp-positivecells were detected in the peripheral blood as determined by FACSand PCR

These two patients are disease-free (clinical CR) and in goodcondition now No side effects associated with the transplantation

of the MDR1-transduced cells were observed No symptom of

lympho/myeloproliferative disease has been observed These results

suggest the safety and feasibility of our MDR1 gene therapy.

279 Retroviral Vector-Mediated In Vivo Transduction of Hematopoietic Stem Cells with Neonatal IV Injection

Lingfei Xu,1 Mark S Sands,2 Alex A Hofling,2 Katherine P.Ponder.1

1 Internal Medicine, Washington University School of Medicine, St Louis, MO, United States; 2 Internal Medicine and Genetics, Washington University School of Medicine, St Loius, MO, United States.

The purpose of this study was to determine if a Moloney murineleukemia-based retroviral vector (RV) could transduce hematopoietic

cells in vivo after neonatal IV injection Homozygous C57BL/6bm1

mice (n=5) were injected IV with 1x1010 transducing units (TU)/kg

of an RV expressing canine Factor IX (cFIX) at 2 or 3 days afterbirth These mice expressed cFIX at 154% of normal levels, and theliver was the major organ transduced with 1.09+/-0.06 (SEM) copies

of the RV per cell at 10 months after transduction as determined byreal-time PCR At 15 months after transduction, peripheral bloodcells from these mice contained 0.024+/-0.006 copies/cell of RVDNA To further test if hematopoietic stem cells were transduced,bone marrow (BM) was harvested from three RV-treated mice at 18months after transduction Unfractionated peripheral blood cellscontained 0.03+/-0.01 copies/per cell, while unfractionated BM cellscontained 0.04+/-0.01 copies/cell of RV DNA FACS-purified BMcells that were Gr1/Mac1-positive (granulocytes and monocytes),B220-positive (B lymophocytes), and TCR-positive (Tlymphocytes) contained 0.06+/-0.03, 0.07+/-0.03 and 0.07+/-0.02copies/cell of RV DNA, respectively (Table 1) Samples from non-transduced mice did not have any detectable RV DNA sequences.Part of the BM from each mouse was used for bone marrow

same colony were used as recipients to enable us to determine ifengraftment occurred by staining for β-glucuronidase activity Two-month-old MPS VII mice were irradiated with 1000 rads before

injected via the tail vein into each recipient The peripheral bloodfrom the recipients contained 0.076+/-0.022 copies/cell of RV DNA

at 3 months after BMT (n=7) (Table 1) We conclude that IV injection

of RV into neonatal mice can transduce pluripotent hematopoietic

stem cells in vivo This could be a problem for liver-directed gene

therapy for hemophilia, since unnecessary transduction of BM cellswould increase the risk of insertional mutagenesis In this study,transduced cells did not appear to have a selective advantage, asperipheral blood cells from hemophilia B mice that were injectedwith the same dose of RV as neonates had a similar copy number of0.03+/-0.01 copies/cell at 6 months after transduction (n=14),although additional studies are necessary to address this possibility

On the other hand, transduction of pluripotent stem cells after IV

Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts S111

injection into newborns might be an effective way to get genes into

BM cells, particularly in large animals where in vitro transduction

has been inefficient

RV DNA copy per cell in the blood and bone marrow

RV DNA copies per cell in donors RV DNA copies per

@18m after RV-transduction cell in recipients

@3m after BMT Peripheral Whole Gr1/Mac1 B220 TCR Peripheral

Blood BM Positive BM Positive Positive Blood

C1 0.01 0.04 0.04 0.05 0.03 0.03+/-0.00 (n=2)

C5 0.04 0.05 0.12 0.13 0.09 0.13+/-0.03 (n=3)

C6 0.04 0.02 0.03 0.03 0.09 0.05+/-0.01 (n=2)

280 Transduction of Primary Human

Macrophages and Dentritic Cells by Recombinant

SV40 Vectors

Pierre Cordelier, Sandra A Calarota, David S Strayer

Monocyte-derived macrophages (MDM) and dentritic cells (DC)

are unique targets in the genetic therapy of infectious diseases and

cancer Gene transfer to MDM and DC has been difficult to achieve

using conventional vector systems because these cells are terminally

differentiated and non-dividing, and their phagocytic functions lead

to degradation of the phagocyted vector particles Recombinant

SV40-derived vectors (rSV40) generally transduce resting cells

efficiently In addition, rSV40 enter cells via caveolae and are then

transported to the nucleus via microtubules, bypassing the phagocytic

process Because of these properties of SV40 vectors, we tested

whether they could transduce MDM and DC effectively For this

purpose, we used two different rSV40s: SV(nef/FLAG), in which

the HIV-1 protein Nef carrying a carboxyl terminal FLAG epitope

is driven by the CMV promoter This construct was used as a

marker, to facilitate detection of effective gene delivery by

immunostaining for FLAG We also used SV(2C7), which carries a

single chain Fv antibody against the cell membrane CCR5 In

SV(2C7), transgene expression is also driven by the CMV promoter

DC and MDM were prepared from buffy coat peripheral blood

monocytes Monocytes were isolated by magnetic sorting for CD11b

These cells were cultured with GM-CSF and M-CSF for two weeks

to produce MDM DC were prepared by sorting for CD14, and

were induced to mature using TNF-a MDM were treated with

either SV(Nef/FLAG) or SV(2C7) Control MDM were

mock-transduced or mock-transduced with SV(HBS), a control rSV40 carrying

the hepatitis B surface antigen Expression of Nef/FLAG and 2C7

was assessed by immunostaining MDM expressed high levels of

these transgenes after transduction with SV(Nef/FLAG) and

SV(2C7), respectively We also tested the ability of rSV40 gene

delivery to blood monocytes to survive the differentiation process

into MDM Thus, peripheral blood monocytes received SV(Nef/

FLAG), SV(2C7), or control treatments, were induced to differentiate

into macrophages as described, then tested for transgene expression

We found that transgene expression continued to be strong in

macrophages derived from rSV40-transduced monocytes In parallel,

we tested the ability of rSV40 vectors to transduce primary DC, by

immunostaining We observed that SV(Nef/FLAG) transduced both

immature and mature DC cells with high efficiency Therefore,

primary human MDM and DC and their more differentiated progeny

may be efficiently transduced with rSV40 vectors These findings

have important implications for approaches to immunization and

treatment of many diseases that entail the ability to deliver transgenes

to monocytes, macrophages and dentritic cells

281 Engineering Hematopoieitc Bone Marrow Cells Resistant to Thymidylate Synthase-Directed Cytoxic Inhibitors

Marg Pena,1 Erin Morrey,2 H Trent Spencer.2

1 Biological Sciences, University of South Carolina, Columbia, SC, United States; 2 Pediatrics; Division of Hematology/Oncology and BMT, Emory University School of Medicine, Atlanta, GA, United States.

The generation of genetically engineered drug resistanthematopoietic bone marrow cells can be used to decrease themyelosuppressive effects of anti-neoplastic chemotherapy agentsand to preferentially increase the percentage of circulating geneticallymodified cells We generated MSCV-based retroviral constructs thatencode the E coli thymidylate synthase (TS) enzyme and showthat the cDNA optimized for expression in mammalian cells(optecTS) confers extremely high-level resistance against TS-directedantifolates Resistance can be conferred to several mouse and humancell lines as well as mouse hematopoietic progenitor cells Attempts

to use the high-degree of resistance for in vivo protection wereunsuccessful in mouse studies due to complications arising fromcirculating thymidine and folate levels and differences in the binding

of inhibitors to mouse transporter proteins compared to humantransporters However, it is shown that gene-modified cells can beselectively enriched ex vivo Similar to our earlier studies, transduction

of mouse bone marrow cells with retroviral vectors encoding bothoptecTS and the green fluorescent protein confers resistance toBW1843U89 and raltitrexed at concentrations that completelyinhibit growth of non-transduced cells, and all transduced cellsgrowing in the presence of inhibitor are brightly fluorescent green,demonstrating that the proviral sequence confers drug resistance.Also, mixing gene-modified mouse bone marrow cells with 70%non-modified cells and plating the mixture in methylcellulose with

or without BW1843U89 selection, the number of GFP positivecolonies are enriched from 20% to >75% In addition, hematopoieticstem cell protection was demonstrated using a competitiverepopulation assay Bone marrow was harvested from 5-FU treatedHW80 mice and co-cultured with MSCV/optecTS producer cellsfor three days On day 3 transduced cells were mixed with an equalnumber of mock transduced C57Bl marrow and BW1843U89 with

or without the nucleoside transport inhibitor dipyridamole Themixed population of cells were selected on days 3, 4 and 5 On day

5 cells were washed and transplanted into a WWv recipient Eightweeks post transplant hemoglobin electrophoresis confirmed, i)optecTS protects hematopoietic stem and progenitor cells fromBW1843U89 induced toxicity and ii) nucleoside transport inhibition

is necessary for selection of primitive hematopoietic cells Theseresults further show that the optecTS construct is a viable markerfor selection of genetically engineered hematopoietic cells

282 T Cells Transfected with Ad-hSSTR2 into T Cells from hCARxTg71xGFP Triple Transgenic Mice Can Be Used To Determine T-Cell Activation and AICD In Vitro and In Vivo

Huang-Ge Zhang,1,2 James M Mountz,3 Qi Wu,1 PingAr Yang,1

Hui-Chen Hsu,1 John D Mountz.1,2

1 Medicine, University of Alabama at Birmingham, Birmingham,

AL, United States; 2 Birmingham VAMC, Birmingham, AL, United States; 3 Nuclear Medicine, University of Alabama at Birmingham, Birmingham, AL, United States.

expresses high levels of a TCR that reacts with the Db/H-Y maleantigen These T cells are autoreactive in H2b male mice but not H2b

female mice, and tolerance is induced by clonal deletion and

down-regulation of CD8 To study these tolerance processes in vivo, we

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S112

have crossed Tg71 mice with GFP transgenic mice and hCAR

transgenic mice to produce triple transgenic mice The T cells were

transfected with an Ad-hSSTR2 that results in expression of high

levels of the human somatostatin receptor (hSSTR2) The FasL and

the T cells with an AdsFas and/or an AdsTNFRI The distribution

of transfered cells expressing the hSSTR2 was determined in vitro

and in vivo using Tc-99m labeled peptides specific for hSSTR2

were first transfected with Ad-hSSTR2 and then transferred into

either male or female H2b mice The initial distribution of T cells to

the spleen was equivalent in male and female mice as determined by

Tc-99m labeled hSSTR2 binding peptides FACS and fluorescent

microscopy analysis of spleen T cells confirmed the initial

distribution In H2bmale, but not female mice, there was an increase

of Db/HY reactive T cells in the spleen on days 2-4 after transfer

followed by apoptosis on days 4-6 after transfer AICD was partially

inhibited in T cells transfected with either AdsFas or AdsTNFRI

separately, but was ablated by transfection with both of the Ad

constructs These results indicate: (i) unstimulated T cells from

hCARxTg71xGFP transgenic mice can be transfected with high

efficiency with an Ad vector, (ii) Ad-hSSTR2 transfer of a nuclear

imaging receptor can be used to follow the homing of autoreactive T

cells in vivo, and (iii) transfer of T cells from Db/H-Y female mice

into H2b male mice results in initial stimulation followed by induction

of AICD and tolerance within one week of transfer AICD was

dependent on both FasL and TNFα signaling These results indicate

the use of ex-vivo cell-gene therapy can be used to assess in vivo

activation and AICD of autoreactive T cells

283 Mobilization of Hematopoietic Stem Cells

and Progenitor Cells to Lung by Intratracheal

Administration of an Adenovirus Encoding

Stromal Cell-Derived Factor-1

Hiroyasu Kobayashi,1 Minoru Tahara,1 Stefan Worgall,1 Shahin

Rafii,1 Ronald G Crystal.1

1 Weill Medical College of Cornell University, New York, NY.

Stromal cell-derived factor-1 (SDF-1), a potent chemoattractant

for lymphocytes and monocytes, also functions to mobilize

hematopoietic stem cells (HSC) and progenitor cells from bone

marrow We have recently demonstrated that HSC and progenitor

cells could be mobilized to the peripheral circulation following

intravenous administration of an adenovirus vector (Ad) encoding

SDF-1 (AdSDF-1; Hattori et al, Blood 2001; 97: 3354-3360) In

this context, we hypothesized that administration of AdSDF-1 to

the respiratory epithelial surface could lead to the establishment of

a SDF-1 gradient between the lung and the peripheral circulation,

and thus attract HSC to the lung To assess this, AdSDF-1 was

administered intratracheally to C57Bl/6 mice at a dose of 1011 particle

units (pu) with administration of an equal dose of AdNull serving as

a control SDF-1 levels were measured by ELISA in the epithelial

lining fluid recovered by bronchoalveolar lavage following vector

administration The lavage fluid levels were highest on day 5 in the

mice treated with AdSDF-1 (2212 ± 315 pg/ml) and significantly

elevated compared to AdNull-treated mice (88 ± 10 pg/ml, p<0.01)

The numbers of cells recovered by lavage from animals treated with

AdSDF-1 also peaked on day 5 (5.3 ± 0.6 x105, p<0.05 compared to

AdNull) To evaluate if HSC were present, the recovered cells were

analyzed by flow cytometry using antibodies against CD34 and

Sca-1 to detect CD34+ Sca-1+ HSC CD34+ and Sca-1+ cells were

significantly elevated in mice receiving intratracheal AdSDF-1 with

the highest levels observed at day 7 (CD34+ 3.5%, Sca-1+ 48%)

and were elevated compared to AdNull (CD34+ 1.9%, Sca-1+ 8.5%)

treated mice To assess if the hematopoietic progenitor cells recovered

by lavage could form colonies, the cells were plated in culture dishes

for 2 hr and the numbers of colonies were counted 14 days later Anaverage of 49 colonies/105 lavage cells was seen with cells fromAdSDF-1-treated mice, whereas no colonies were detected withcells derived from AdNull treated mice To evaluate if the cellsrecovered by lavage of the AdSDF-1-treated mice had marrowrepopulating capacity, washed lavage cells (5x105/mouse) recovered

7 days following administration with AdSDF-1 were administeredintravenously to lethally irradiated syngeneic mice The micereceiving lavage cells from AdSDF-1-treated mice survivedsignificantly longer than animals who had received lavage cells fromAdNull treated mice (p<0.05), suggesting that the SDF-1 recruitedpopulation contains cells with stem cell repopulating capacity Thisdata suggests that local administration of AdSDF-1 to the respiratorytract can induce the mobilization of HSC and progenitor cells withrepopulating capacity to the lung This approach could serve as abasis for new strategies to study stem cell recruitment to the lungand for the development of novel therapies using HSC and progenitorcells attracted to the respiratory tract

Dr Crystal has equity in, is a consultant to, and receives sponsoredresearch funds from, GenVec, Inc., Gaithersburg, Maryland, apublicly-traded biotechnology company

284 Long-Term Follow-Up of Dogs and Baboons Transplanted with CD34+ Cells Transduced by Oncoretroviral or Lentiviral Vectors

Julia C Morris,1 Bobbie M Thomasson,1 Laura J Peterson,1

Peter A Horn,1 Tobias A Neff,1 Robert G Andrews,1,2 Christofvon Kalle,3 Robert E Richard,4 C Anthony Blau,4 MartinGoerner,1 Michael Harkey,1 Peter Kurre,1,2 Hans-Peter Kiem.1,4

1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 2 Department of Pediatrics, University of Washington School of Medicine, Seattle, WA; 3 Experimental Hematology, Children’s Hospital Medical Center Cincinnati, Cincinnati, OH; 4 Department of Medicine, University of Washington School of Medicine, Seattle, WA.

The recent report of the development of T-cell leukemia in twochildren treated for X-SCID with gene therapy in France hasprompted us to review gene-marking levels and clinical outcomes inour large animal gene therapy studies in dogs and baboons Recentimprovements in stem cell transduction protocols have led tosubstantial increases in gene-marking levels in large animals Wenow almost routinely observe therapeutically relevant marking levels

of greater than 5% of peripheral blood leukocytes and up to 30% insome animals Given the high numbers of gene-marked cells infusedinto our animals, one would predict that we would have seenoutgrowth of a gene-modified clone or leukemia in some of ouranimals if the development of leukemia in two of the 11 childrentransplanted in the X-SCID study was due solely to insertionalmutagenesis To date, we have reviewed data from 42 animals, 15dogs and 27 baboons, with a follow-up >6 months and markinglevels >1% All animals received CD34+ marrow or peripheral bloodcells transduced with oncoretroviral and/or lentiviral vectorsfollowing myeloablation The oncoretroviral vectors used in thesestudies were MoMuLV-derived vectors encoding neomycinphosphotransferase (neo), enhanced green fluorescent protein(EGFP) or its yellow variant EYFP or the selectable markersF36Vmpl or the p140k mutant of methylguanine DNAmethyltransferase (MGMT) The lentiviral vectors used weresecond-generation SIN vectors encoding EGFP or EYFP Thetransduction culture conditions included CH-296 and multiple growthfactors (IL3, IL6, SCF, G-CSF, FLT3-L and MGDF) The mediannumber of cells infused was 7.5 x 106 cells/kg for the baboons (range1.4-30.9 x 106/kg) and 13.0 x 106 cells/kg for the dogs (range 1.9-74.0 x 106/kg) Follow-up ranged from 158 to 2007 days with a

Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts S113

median of 639 days The frequency of gene-marked cells in peripheral

blood and marrow leukocytes, red cells and platelets was monitored

at regular intervals by flow cytometry and PCR and complete blood

counts were obtained at the time of each blood draw Although in

some of the animals gene-marking increased transiently after infusion

of gene-marked cells or after administration of a selective agent (a

chemical inducer of dimerization), no obvious clonal outgrowth was

observed based on flow cytometric analysis of multiple

hematopoietic lineages None of the animals that died or were

euthanized at the end of the study showed any evidence of leukemia

or a myeloproliferative syndrome We are currently performing

clonality analyses in animals with high-level marking to determine

clonal fluctuation over time and to rule out the development of oligo

or monoclonality In summary, we have seen no evidence of leukemia

after transplantation of gene-modified cells in any of our animals

The follow-up presented here (median 1.75 years) is comparable to

3 to 5 years in children given the shorter life span of dogs and

baboons Further analysis of the clonal engraftment pattern after

transplantation will be presented at the meeting

285 Genetically Engineered Autologous

Marrow Stromal Cells Sequestered within a

Human-Compatible Bovine Collagen Matrix for

Prolonged and Reversible In Vivo Systemic

Delivery of Functional Erythropoietin in Mice

Nicoletta Eliopoulos,1 Laurence Lejeune,1 Daniel Martineau,2

Jacques Galipeau.1,3

1 Lady Davis Institute for Medical Research, McGill University,

Montreal, QC, Canada; 2 Department of Veterinary Medicine,

Université de Montréal, St-Hyacinthe, QC, Canada; 3 Division of

Hematology/Oncology, Jewish General Hospital, McGill

University, Montreal, QC, Canada.

Bone marrow stromal cells (MSCs) can be exploited

therapeutically in transgenic cell therapy approaches These

autologous cells, with their strong proliferative ability, can be

genetically engineered into synthetic endocrine cells, expanded in

sufficient number for clinical effect, and returned to the donor

However, should unanticipated complications result from use of

gene-modified cells, it would be impossible to remove the MSCs if

introduced by intravenous or intraperitoneal injection Consequently,

the capacity to deliver these cells subcutaneously (s.c.) as a

retrievable implant would augment the safety of this strategy

Therefore, the aim of this study was to determine if gene-modified

MSCs sequestered within the FDA-approved, bovine type I

collagen-based material Contigen™(Bard Canada) can serve as a

retrievable implant for systemic delivery of functional erythropoietin

(Epo) when injected s.c in normal immunocompetent mice We

generated a monocistronic retroviral construct comprising murine

Epo cDNA, stably transfected GP+E86 packaging cells, and

employed retroparticles to transduce primary MSCs from C57Bl/6

mice A clone of Epo gene-modified MSCs, revealed by ELISA to

secrete 3 Units of Epo per 106 cells per 24 hrs, was implanted at 107

MSCs per syngeneic mouse, s.c (1) without a matrix and (2) mixed

in Contigen In 5 mice implanted with these MSCs without a matrix,

the hematocrit (Hct) increased from 57 ± 0.7% (mean ± SEM) prior

to implantation, to a peak 70 ± 3.2% at ~3 weeks following

implantation, and gradually dropped to a basal 57 ± 2.4% at 7

weeks In contrast, in mice (n=5) implanted with Contigen-embedded

MSCs, the Hct rose from 51 ± 0.2% pre-implantation, to 81 ± 0.9%

at ~3 weeks post-implantation, and further climbed, maintaining

levels of 82-88% until week 15 and >70% up to ~29 weeks ensuing

implantation (p<0.0001 Logrank) Plasma Epo concentration in these

mice rose from 2.5 ± 0.4mU/ml pre-implantation to 30-50mU/ml

commencing 2 weeks post-implantation and descending to ~15mU/

ml at week 16 In a separate experiment to ascertain the implant

retrievability safety feature of the approach, 9 mice were implanteds.c with Contigen-embedded Epo-secreting MSCs and ~3 weekslater, implants removed from 4 recipients The Hct in these 4 micedecreased from 77 ± 2.7% at 3 weeks post-implantation to baselinelevels of 55 ± 1.2% 2 weeks following implant harvesting, whereas

in mice with implant left intact, the Hcts at these time points were

76 ± 2.7 and 80 ± 2.9% Control mice implanted with Contigen only

or Contigen with green fluorescent protein (GFP) gene-modifiedMSCs had Hcts unchanged from baseline Flow cytometry analysisrevealed GFP-engineered MSCs as mainly CD34-, CD31-, CD45-and CD44+, both prior to and ~3 weeks succeeding implantationmixed in Contigen In conclusion, this investigation demonstratesthat s.c implanted MSCs embedded in human-compatible matrixContigen offers a safe reversible approach for delivery of plasmasoluble therapeutic proteins, in addition to a more persistentpharmacological effect as compared to non-embedded MSCs

286 Gene Delivery to Primary Peripheral Blood Lymphocytes: Efficient and Effective Transduction of PBL with Single and Multiple rSV40 Vectors

David S Strayer,1 F Branco.1

1 Pathology, Anatomy and Cell Biology, Jefferson Medical College, Philadelphia, PA.

Background: Gene delivery to primary blood cells, particularlylymphocytes, has been difficult to achieve Most approaches require

or prefer lymphocyte stimulation, are generally inefficient andconstrain practical gene delivery to lymphocytes to the small

percentages of the total pbl pool that can be handled ex vivo The

inefficiencies of single gene delivery to pbl are compounded if onetries to deliver multiple genes Since recombinant SV40-derivedvectors (rSV40s) transduce unstimulated, unselected, pblpermanently and at high efficiency we tested whether these vectorscould deliver multiple genes in sequence to pbl We further askedsequential rSV40 combination gene therapy improved functionality.Methods: Normal human buffy coats were separated using ficoll,and then depleted of monocytes by plastic adherence Resultingpbl-enriched populations were transduced without stimulation orselection using one or more rSV40s carrying anti-HIV transgenes.After transduction, cells were cultured with IL-2 and challengedwith HIV-1NL4-3 at virus:cell ratios (MOI) of 0.005, 0.01, 0.015and 0.03 Transgene functionality was measured as inhibition ofHIV replication, as assayed by ELISA for supernatant HIV p24.The rSV40s used carried: RT3 and IE8, single chain Fv antibodies(SFv) to HIV reverse transcriptase; Aw, a SFv to HIV integrase; HE,

a SFv against CXCR4; RevM10, a dominant negative mutant ofHIV Rev; PolyTAR, a polymeric TAR decoy; and (as a control)HBS, hepatitis B surface antigen

Results: Previous studies had documented simultaneousexpression of several transgenes by >95% of unselected cells aftersequential rSV40 transduction Expression of the transgenes usedhere was demonstrated by Western and Northern blotting,immunostaining and flow cytometry Effectiveness of SV(HE) genedelivery was tested by FACS analysis, showing decreased cellmembrane CXCR4 Singly- and multiply-transduced cells werechallenged with progressively higher doses of HIV It is characteristic

of gene delivery to inhibit HIV that protection of transduced cellsdeteriorates as challenge doses of HIV increase Thus, most individualtransgenes protected pbl completely from HIV challenge at MOI =0.005, but protected only partially or not at all at higher doses.Only SV(HE), which decreases CXCR4, protected singly at thehighest HIV dose, MOI = 0.03 Sequentially delivered combinations

of 2 or 3 transgenes generally protected better than any componenttransgene did alone Some combinations, e.g., Aw (anti-IN SFv) +

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IE8 (anti-RT SFv), protected well at challenge HIV doses that

overwhelmed either alone The most effective combinations used

SV(HE) or SV(PolyTAR) together with other transgenes

Conclusions: Primary blood lymphocytes can be easily

transduced with rSV40 vectors to >95% efficiency without selection

Delivery of multiple mutually supporting transgenes, such as the

HIV-inhibitory genes used here, may improve functionality as

compared with single genes Finally, the high titers (>1012/ml) and

>95% transduction efficiency without selection that are characteristic

of rSV40 vectors facilitate combinatorial gene delivery to pbl and

other blood cells

287 Electroporation of Non-Human Primate

with Erythropoeitin Plasmid DNA Results in

Sustained High Hematocrit with No Adverse

Effects

Adrian Vilalta,1 Michal Margalith,1 Suezanne Parker,1 Michael

Sawdey,1 May de las Alas,2 Robert Bernard,2 Drew Hannaman.2

1 Gene Optimization and Design, Vical Inc, San Diego, CA, United

States; 2 Ichor Medical Systems Inc, San Diego, CA, United States.

Data from rat and rabbit studies have shown that a sustained high

level of erythropoeitin (EPO) expression can be obtained after

intramuscular injection of plasmid DNA followed by electroporation

Circulating levels of EPO in the rat after DNA injection followed by

electroporation were nearly 100 fold higher than those obtained

with plasmid DNA injection alone Data from rat and rabbit studies

were used to design a non-human primate study where we examined

the efficacy and safety of delivering EPO-encoding plasmid DNA

by intramuscular injection followed by electroporation Six rhesus

monkeys were injected with rhesus EPO-expressing plasmid

followed by electroporation Monkeys were electroporated using

the TriGrid electrode array system and TGP-2 pulse generator

(Ichor) Two control animals were injected with the plasmid DNA

but were not electroporated EPO serum levels, hematocrit as well

as serum chemistry and a full panel of hematological parameters

were followed for over 60 days after administration Data from

electroporated monkeys indicated that circulating EPO reached a

at day 35 Elevated hematocrit was maintained until the termination

of the study, 120 days after plasmid administration No changes in

either circulating EPO or hematocrit were evident in the

non-electroporated group Treatment of non-human primates with

plasmid DNA followed by electroporation appeared safe with no

abnormalities in serum chemistry detected EPO levels obtained

through plasmid DNA injection / electroporation are well within the

therapeutic range for this protein and are comparable to published

results obtained in the same animal system with adenoviral delivery

Results from the non-human primate study suggest that

intramuscular injection of EPO plasmid DNA followed by

electroporation can be successfully scaled up from small mammal

models and may provide an effective therapy for patients with

Christian Chabannon,1,3 Frederic Viret,2 Anne-Marie Imbert,1

Dominique Genre,2 Didier Blaise,3 Dominique Maraninchi,2

Patrice Viens.2

1 Centre de Therapie Cellulaire et Genique, Institut Calmettes, Marseille, France; 2 Departement de Medecine, Institut Paoli-Calmettes, Marseille, France; 3 Departement d’Onco- Hematologie, Institut Paoli-Calmettes, Marseille, France.

Paoli-From 1999 through 2000, six patients received geneticallyengineered cells, along with unmanipulated autologous peripheralblood cells and progenitors to support the administration of high-dose chemotherapy for poor-risk breast or ovarian cancer (Bagnis et

al, Exp Hematol, 2002) Peripheral blood CD34+ cells were obtained

by immuno-selection from autologous aphereses, and were engineered

in vitro to express a modified version of E coliβ-galactosidase, using

a Moloney-derived retroviral vector We reported low level andtransient expression of the marker gene in peripheral blood and bonemarrow cells obtained during the first three months following infusion.Recently, two cases of clonal T-cell proliferations were reported

among 11 children who were treated for X-SCID, using ex vivo gene

transfer of the γc receptor chain in bone marrow CD34+ cells; inboth cases, the retroviral vector integrated near the LMO2 locus.These severe adverse events were reported after the description ofleukemia in a murine model of marker gene transfer in bone marrowcells Insertional mutagenesis is suspected as the mechanism thatinduced cell proliferation

These observations prompted us to offer our patients additionalfollow-up and counselling Of six patients, three had since died, atdays 616, 671 and 249 after autologous transplantation respectively,

as a result of their initial tumor progression; medical records contained

no indication that they had in addition developed any hematologicalabnormality that could be related to genetic manipulation of theirprogenitor cells Three patients were alive at days 1069, 935 and

728 after transplantation, and were seen again in the outpatientclinic Clinical examination and blood counts revealed nohematological abnormality β -galactosidase activity was studied byimmunocytochemistry (X-gal), and genomic integration of theretroviral vector was studied with PCR, on blood mononuclear cells:all three samples were negative with both techniques An additionalpatient died shortly after these investigations, at day 1109 afterinfusion of genetically modified cells, still showing no hematologicalabnormality that could be attributed to the procedure This suggeststhat the progeny of genetically modified cells has disappeared inthese patients, or persist at very low and undetectable levels.Gene marking in adult cancer patients represents a differentsituation from gene therapy of X-SCID in children Geneticallymodified cells have no survival advantage in this situation Whilecancer patients display some form of immuno-suppression as aresult of chemotherapy, they are however significantly moreimmunocompetent than are children with X-SCID, and thus lessprone to opportunistic infections such as varicella-zoster that maytrigger lymphoid proliferation These factors may contribute to lowerthe risk that a clonal proliferation arrises as the result of insertionalmutagenesis; however, a careful and extended monitoring will benecessary to assess the exact probabilty that such an event mayoccur in cancer patients

Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts S115

289 T Cell Stimulation Using Cytomegalovirus

pp65-Modified Dendritic Cells

Bjorn Carlsson,1 Wing-Shing Cheng,1 Thomas H Totterman,1

1 Clinical Immunology, Rudbeck Laboratory, Uppsala University,

Uppsala, Sweden.

Cytomegalovirus (CMV) infection is a dangerous complication

in immunosuppressed individuals such as allogeneic stem cell

transplant patients CMV disease can be prevented by early

posttransplant transfer of donor-derived CMV-directed T cells Fast

and cost efficient methods to generate CMV-specific T cells are

therefore warranted The current studies utilized peptide-pulsed

and adenovirus-transduced dendritic cells (DCs) to generate

peptide-pulsed DCs and three restimulations with peptide-pulsed

monocytes virtually all T cells were CD8+, expressed the relevant

T cell receptor and exhibited high peptide-specific lytic activity

Already after one stimulation, pp65495-503-restriced T cells could be

sorted to a purity of higher than 95% and expanded up to 1000-fold

in two weeks This technique may prove useful for rapid generation

of large amounts of specific cytolytic T lymphocytes (CTLs) for

cell therapy DCs transduced with an adenoviral vector coding for

the full-length pp65 protein (Adpp65) were able to simultaneously

expand CTLs against multiple epitopes of pp65 In addition, they

activated CMV-specific CD4+ helper T cells This approach would

stimulate multiple-epitope populations of pp65-specific T cells

and could be made available to patients of any HLA haplotype

DCs transduced with adenoviral vectors to express full-length

antigens may prove to be potent vaccines against viral pathogens

and cancer

290 Ectopic Telomerase Expression Elongates

Telomeres While Maintaining Osteogenic

Potential of Bone Marrow Stromal Cells

Nedime Serakinci,1 Thomas G Jensen,1 Moustapha Kassem.2

1 Human Genetics, University of Aarhus, Aarhus C, Denmark;

2 Human Genetics, University of Aarhus, Aarhus C, Denmark;

3 Endocrinology, Odense University Hospital, Odense C,

Denmark.

Expression of the catalytic subunit of human telomerase (hTERT)

into certain primary human cell types prolongs the life span We

used retroviral transduction of human bone marrow stromal cells

(hMSC) to develop ectopic telomerase expressing mesenchymal

cell line as a model to investigate telomerase regulation and

tumorigenicity of human cells (Nat Biotechnol 20:592-596; 2002)

The cell line can differentiate into multiple cell types including

osteoblasts, adipocytes, chondrocytes, myocytes and possibly other

cell types It has been growing in our labs for more than 3 years and

has undergone around 350 population doublings At the same time

it is able to differentiate and express functional cell markers at a level

similar to early passage normal cells When transplanted in vivo in

immunodeficient mouse, it forms bone in a magnitude that is larger

than that formed by an equal number of normal cells

The cell line is attractive for drug testing and screening before

animal studies It can be employed in a wide range of applications

including drugs with potential in enhancing bone formation, cartilage

formation or inhibiting adipocyte formation or enhancing the

functional abilities of the cells It may at a later stage be used for

therapeutic purposes for the treatment of osteoporosis, bone fracture

etc and to produce medical relevant proteins (growth factors;

hormones) relevant in a variety of diseases

The cells maintained genomic instability and did not form tumor

when subcutaneously injected in immune-deficient mice and had a

normal karyotype Telomere lengths in individual chromosome arm

and chromatids were detected and sized with the dideoxy-Primed InSitu Labeling (ddPRINS) method (Nat Biotechnol.17: 200-201; 1999)and computer assisted telomere quantifier soft wear (DAKO,Denmark) During continuous in vitro subculturing, telomere lengthdistribution in early passages showed uneven distribution until PD128telomere lengths became stable and show an even distribution.The immortal mesenchymal cells expressing telomerase can beused to study mechanisms behind the induction of neoplasia andeffects of telomerase inhibition

291 Towards Gene Transfer Using a Model of Autologous Haemopoietic Stem Cell

Transplantation with Nonmyeloablative Conditioning in Baboons

S Larsen,14 M Jackson,4 V Patel,4 M Haque,4 L Duke,3 S.Thomson,4 K Chng,1 M Armstrong,4 J Gibson,4 A Hennessy,4

Engraftment of therapeutic haemopoietic stem cells (HSC) requiressome form of conditioning This is particularly the case for gene-modified cells that lack a selective survival advantage To avoid thetoxicity of myeloablative conditioning, several groups have examinedthe use of nonmyeloablative conditioning with different species,radiation doses and energies Although toxicity has been low, thelevel of gene marking has generally been below levels of therapeuticutility (0.01% to 15%) To explore the use of nonmyeloablativeconditioning, we are developing a baboon (Papio hamadryas) model

of HSC gene transfer using 600cGy of megavoltage x-rays We haveperformed 7 mobilizations (4 with G-CSF 100mcg/kg/d and 3 withG-CSF 100mcg/kg + SCF 50mcg/kg/d) followed by leukapheresis

on day 5 using the Cobe Spectra Apheresis System (n=6 animals).Owing to initial difficulties with vascular access and inadequateanticoagulation, we now use an 18G arterial outflow catheter in thefemoral artery and anticoagulate with ACDA and heparin All animalstolerated general anaesthesia without side effects The results suggestthat G-CSF + SCF is more effective than G-CSF alone in mobilizingCD34 cells with 27-64 CD34+ cells/uL vs 11-28 CD34+ cells/uL inthe peripheral blood on day 5 and 4.2-13 x 10e6/kg vs 0.45-0.6 x10e6/kg total CD34 cells in the leukapheresis product respectively

To assess safety and feasibility, we have performed one autologoustransplant using non-marked cells Peripheral blood mononuclearcells were harvested on day 5 of G-CSF + SCF mobilization Theleukapheresis product containing 4.2 x 10e6/kg CD34+ cells wasfrozen and stored in liquid nitrogen After modelling radiationdosimetry based on CTScans, the 21kg animal was irradiated using

a 6 MV photon beam on a Varian Clinac6 linear accelerator A dose

of 600 cGy was delivered to the mid-plane in 75 minutes usingbilateral fields at a dose rate of 8 cGy per minute Thermo luminescentdosimeters were used to measure the actual dose delivered Theaverage deviation from the intended dose was 1 % and the measuredmid-plane dose was 1.6% lower than prescribed The CD34+ cellswere thawed and infused 48 hours after the irradiation The animaltolerated the procedure well, although an infection occurred duringwhite cell recovery which resolved with antibiotic treatment.Transient pancytopenia was observed with a neutrophil nadir of0.6 x 10e9/L on day 8, platelet nadir of 18 x 10e9/L on day 10 andrecovery of all cell counts by day 12 post-irradiation In establishing

a nonmyeloablative primate model of autologous haemopoietic stemcell transplantation we have examined several variables includingmobilization regimens, radiation dosimetry and safety In light ofthe recent report that G-CSF + SCF mobilized cells are more efficiently

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transduced than G-CSF-alone or G-CSF + Flt3L mobilized cells

(Hematti et al, Blood, 2002 online) and our results showing more

efficient mobilization using G-CSF + SCF, we conclude that this

combination may be optimal in the context of haemopoietic stem

cell gene transfer protocols

292 Application of a Human Multidrug

Transporter (ABCG2) Variant as Selectable Marker

in Gene Transfer to Heamatopoietic Progenitor

Cells

Olga Ujhelly,1 Csilla Özvegy,2 György Várady,1 Balázs Sarkadi,1

Katalin Német.1

1 National Medical Center, Institute of Haematology, Budapest,

Hungary; 2 Institute of Enzymology, Hungarian Academy of

Sciences, Budapest, Hungary.

Stem cell based gene therapy is often unsuccessful because of the

relatively low number of genetically modified cells with repopulating

capabilities In order to provide a selective advantage for the modified

cells we applied the human ABCG2 protein, a resident xenobiotic

transporter in stem cells as a selectable marker This protein is

active as a homodimer, and its relatively small cDNA is an advantage

in gene therapy applications In the present study a mutant form of

ABCG2 (R482G), showing drug-pumping activity with an altered

substrate-specificity was co-expressed with a therapeutic gene by

using a bicistronic vector and an efficient retroviral transduction

protocol Expression of the gp91phox protein in human gp91phox

-knock-out hematopoietic progenitor cells corrected the

loss-of-function mutation responsible for human chronic granulomatous

disease, while the mutant ABCG2 protein selectively protected the

transduced cells against clinically applicable cytotoxic agents

Overexpression of ABCG2 did not affect hematopoietic cell

maturation or the restoration of granulocyte function by gp91phox

We suggest that the mutant ABCG2 protein is an ideal candidate for

human stem cell protection and for use as a selectable marker in gene

therapy

293 Protection of the Hematopoietic Stem Cell

Compartment by Retroviral Gene Transfer of Drug

Resistance Genes

Thomas D Southgate,1 Lorna B Woolford,1 Claire F Stevens,1

Leslie J Fairbairn.1

1 Gene Therapy, Paterson Institute for Cancer Research,

Manchester, United Kingdom.

Even after high-dose chemotherapy followed by autologous

transplantation with peripheral blood-derived progenitor cells,

relapse of metastatic tumours still constitutes an important problem

Elimination of residual malignant cells will require the practice of a

consolidation treatment but with many chemotherapeutic agents

conferring acute bone marrow toxicity there is a need to minimise

side effects whilst allowing effective tumour management

Retrovirally transduced hematopoietic stem cells overexpressing

the ATP-dependent drug efflux pumps, P-glycoprotein or multidrug

resistance associated protein (MRP) may confer chemotherapeutic

protection This could avoid the associated acute bone marrow

toxicity in these patients, and in doing so widen the therapeutic

window for an earlier onset of post-transplantation chemotherapy,

whilst eliminating the risk of leukopenia, anemia and/or

thrombocytopenia which so far jeopardises treatment and can

compromise the patient’s quality of life

Here we present data from a mouse model, that mimics clinically

achievable gene transfer rates, to demonstrate the efficacy of

protection of a minor proportion of the repopulating cells with

ATP-dependent efflux pumps We have utilised novel retroviral

facilitate a direct in vivo competition assay of these two transgenes.

Transplantation of bone marrow expressing both GFP and MDR-IRES-YFP within the same animal allows us todetermine the relative protection conferred to these twohematopoietic populations in response to etoposide and paciltaxelboth in the presence and the absence of the ATP-dependent drugefflux pump inhibitor verapamil Here we compare this protection

MRP-IRES-to determine whether P-glycoprotein or MRP confers greaterchemoprotection against clinically approved chemotherapeuticagents

294 Regression of Murine Bladder Tumors by AdCD40L Therapy

Angelica S I Loskog,1 Thomas H Totterman.1

1 Oncology, Radiology and Clinical Immunology, Clinical Immunology Division, Uppsala, Sweden.

Aim

In previous studies we have shown that s.c.vaccinations withCD40L expressing MB49 tumor cells protect mice from challengewith parental tumor (J.Urol.166:1093, 2001) In this preclinicalstudy we investigated the immunological mechanisms and evaluatedthe capacity of treating established tumors with CD40L immunogenetherapy

BackgroundMany tumors exhibit immune escape properties that enable theirsurvival Murine bladder cancer cells (MB49) induce local IL10production IL10 is known to drive the potent anti-tumor Th1response towards a less favorable Th2 response We have studiedthe immunological effects (cytokine levels) of introducing theCD40L, a potent dendritic cell activation molecule, into the tumormicro milieu by use of ELISA and quantitative PCR Further, westudied induction of MB49 specific CTLs by 51Cr release assays.Finally, human biopsy material was screened for immune escapeproperties by PCR

ResultsAdCD40L injections induced regression of MB49 tumors andfurther, stabilized and suppressed growth of large solid tumors.Fibroblasts or DCs can also be used as carrier cells of the CD40Lgene in order to suppress tumor growth The cytokine pattern ofuntreated tumors were of Th2 type with high IL10 levels Expression

of CD40L in the tumor environment suppressed IL10 productionand favored Th1 cytokine production Further, AdCD40Limmunogene therapy stimulated induction of MB49 specific CTLs.MB49 cells and human biopsies exhibited immune escapemechanisms such as deteriorated FAS expression and high levels ofthe granzyme B inhibitor SPI-6/PI-9 or apoptosis inhibitor cFLIP.Conclusion

In conclusion, despite powerful escape mechanisms such as IL10induction, poor FAS and high SPI-6 expression, AdCD40Limmunogene therapy can effect the regression of s.c tumors Thisstudy further argues for using CD40L immunogene therapy in thetreatment of bladder carcinomas

Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts S117

295 Ad.IFN Beta Therapy Synergizes with

COX-2 Inhibition To Supress Large Lung and

Mesothelioma Tumors in Balb/c Mice

Peter A DeLong,1 Tom Tanaka,1 Robb Kruklitis,1 Larry Kaiser,1

Steve Albelda.1

1 Throcacic Oncology Laboratory, University of Pennsylvania,

Philadelphia, PA, United States.

RATIONALE: We reported success treating early mesothelioma

(MM) and lung carcinoma (LC) with 1e9 plaque forming units (pfu)

of Ad.IFN-b This therapy is ineffective in large tumors To eradicate

advanced tumors, combined therapies may be needed Since MM

and LC over-express cyclooxygenase 2 (COX-2) we studied the

effect of COX-2 inhibition in mice bearing MM and LC tumors, and

examined COX-2 inhibition combined with Ad.IFN-b therapy

METHODS: Balb/c mice were fed chow with and without a

COX-2 inhibitor prior to, or simultaneously with, subcutaneous (s.c.)

injection with mouse MM (AB12) and LC (L1C2) cells The

combination of COX-2 inhibition and Ad.IFN-b therapy was

examined by injecting some mice intratumorally (i.t.) with 1e8 pfu

or 1e9 pfu Ad.IFN-b The mechanism of COX-2 blockade was

investigated by repeating experiments in immunodeficient SCID

mice and mice depleted of CD4+ and CD8+T cells Histology was

examined by freezing and sectioning excised tumors, and staining

them with antibody specific for CD4, CD8 and CD45 The effect of

COX-2 inhibition on the presence of cytotoxic T lymphocytes

(CTLs) was assayed by mixing CD8 + T-cells from treated and

untreated mice with AB12 cells at a ratio of 3:1, and injecting this

mixture into naive mice (Winn Assay) RESULTS: COX-2 inhibition

started prior to, or simultaneous with, tumor injection suppressed

MM and LC tumors, and in small tumors (100mm3) it slowed, but

did not eradicate, these tumors In large tumors (500mm3) COX-2

inhibition had little effect COX-2 inhibition combined with 1e8 pfu

Ad.IFN-b i.t cured small tumors, and combined with 1e9 pfu Ad.IFN

b i.t suppressed large tumors This effect of COX-2 inhibition was

lost in SCID mice CD8+ T-cell depletion eliminated tumor

suppression caused by COX-2 blockade, while CD4+ T-cell

depletion had no effect COX-2 inhibition did not affect the number

or activity of CTLs, however histology demonstrated increased

tumor infiltrating lymphocytes (TILs) in the presence of COX-2

blockade CONCLUSION: COX-2 blockade suppresses tumor

growth in small but not large MM and LC The anti-tumor effect is

dependent on CD8+ T cells, and associated with increased TILs,

but is not due to increased numbers of CTLs COX-2 inhibition and

Ad.IFN-b treatment synergize to suppress the growth of large

mesothelioma and lung cancer tumors

296 Combined Oncolytic and Immuno-Therapy

in Breast Cancer Mouse Models Using Adenoviral

Vectors

Anh-Thu Tieu,1 Kathrin M Bernt,1 Shaoheng Ni,1 Andre Lieber.1

1 Division of Medical Genetics, Dept of Medicine, University of

Washington, Seattle, WA.

Conditionally replicating adenoviral vectors have been used in a

large number of xenograft mouse models of cancer as well as several

clinical studies In these studies, a question that is often not addressed

and still largely un-resolved is the impact of the immune-system on

viral replication and anti-tumor efficacy Anti-viral immune responses

are likely to be responsible for the shut-down of viral replication

observed in clinical studies within a few days after administration

On the other hand, the strong cellular immune response elicited by

replicating vectors might be helpful in breaking tolerance towards

tumor cells in an immuno-therapy setting In this study, we lay the

foundations to test the latter hypothesis in several syngeneic breast

cancer mouse models Specifically, we plan to combine a conditionally

replicating vector expressing a cytotoxic transgene with dendriticcell- and NK-mobilization through Flt3L We propose that infectedand/or apoptotic tumor cells may serve as a source of antigen, whilethe strong inflammatory local cytokine response triggered by theproductive viral infection represents a milieu that is favorable forefficient activation and maturation of antigen presenting cells

We have analyzed five mouse breast cancer cell lines that formtumors in syngeneic hosts, and found that all support viraltransduction, replication and cytopathic effect, albeit to varyingdegrees: EMT-6 was more susceptible than C3L5 and TM40D,which were more susceptible than JC and 4T1 In parallel, we havetested the induction of apoptosis by an E1/E3 deleted vectorexpressing Trail in EMT-6, TM40D, JC and 4T1, and found EMT-

6 and 4T1 to be sensitive We went on to create a conditionallyreplicating variant of this vector, Ad.IR-Trail/E1A, which will beused as oncolytic/cytotoxic modality in our combination treatmentstudies Finally, we have constructed an E1/E3 deleted vector whichexpressed high levels of Flt3L (Ad.Flt3L) Mice injected with thisvector displayed a 7-fold increase in leukocyte count, and a 10-foldincrease in spleen size 10 days after vector administration Leukocytesubsets that were preferentially mobilized comprised myeloid, NK-and dendritic cells We now plan to combine all three treatmentmodalities in vivo, tumor specific viral replication, Trail-inducedapoptosis, and Flt3L-mediated NK-and dendritic cell mobilization.Finally, immuno-competent, syngeneic mouse tumor modelswhich support productive adenoviral replication have not beendescribed so far, and their absence has greatly hampered the researchdirected towards the interplay of viral replication, anti-viral immune-response and anti-tumor effects The models described here willalso be very helpful to address question such as the immune-mediatedtermination of viral replication or the role of the E3 region genes inproductive infection and immune-evasion

297 Anti-Tumor Immunity Against Bladder Cancer Induced by Ex Vivo Expression of CD40 Ligand

Takahiro Kimura,1,2 Toya Ohashi,2 Tetsuro Kikuchi,3 TakehitoNaruoka,1,2 Hiroshi Kiyota,1 Yoshikatsu Eto,2 Yukihiko Ohishi.1

1 Department of Urology, Jikei University School of Medicine, Minato-ku, Tokyo, Japan; 2 Department of Gene Therapy, Jikei University School of Medicine, Minato-ku, Tokyo, Japan;

3 Department of Neurosurgery, Jikei University School of Medicine, Minato-ku, Tokyo, Japan.

Purpose: The interaction between CD40 ligand (CD40L) andCD40 on antigen-presenting cells is essential for the initiation ofantigen-specific T cell responses In order to clarify whetherexpression of CD40L in tumor cells is useful as a new strategy ofsystemic therapy against bladder cancer, we investigated the anti-tumor immunity induced by CD40L in mouse bladder cancer cells,MBT2 Materials and Methods: MBT2 was transduced by theretroviral vector expressing CD40L (MBT2-CD40L) The level ofIL-12 released by mouse bone marrow-derived dendritic cells(BMDCs) co-cultured with MBT2-CD40L cells was determined

by ELISA, to demonstrate the ability of MBT2-CD40L to activatedendritic cells The anti-tumor effects induced by CD40L wereassessed using subcutaneous and orthotopic tumor cell injectionmodels In order to evaluate the efficacy of CD40L expressing onMBT2, MBT2 and MBT2-CD40L cells were injected eithersubcutaneously or intravesicaly For vaccination model, MBT2-CD40L cells were s.c inoculated on days -14 and -7, and MBT2cells were injected into a distant site on day 0 For therapeuticmodel, MBT2 cells were injected on day 0, and MBT2-CD40Lcells were s.c inoculated on days 3 and 10 CTL assay, antibodyablation study and detection of serum antibody against MBT2 weredetermined to elucidate the effector cell fumctions Results: BMDCs

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co-cultured with MBT2-CD40L cells produced IL-12 eight times

more than those co-cultured with parental MBT2 cells Injected

MBT2-CD40L cells were rejected promptly in both subcutaneous

and orthotopic model The vaccination of MBT2-CD40L cells

induced anti-tumor immunity against parental tumors at a distant

site in both subcutaneous and orthotopic model However

anti-tumor effect against preexisting anti-tumors was insufficient by

MBT2-CD40L inoculation Antibody ablation study and detection of serum

for the anti-tumor immunity and serum antibody against MBT2

was produced in vaccination model Conclusions: These data

demonstrated that anti-tumor immunity induced by CD40L was

effective in vaccination model and suggest that the immuno-gene

therapy using CD40L may prevent recurrence of bladder cancer We

thank Dr Kohn (Univ Southern California) for providing us

pMND-x-SN retrovirus vector

298 Treatment of Subcutaneous Melanoma

after Vaccination with Cells Expressing

Xenoepitopes in the αα αα(1,3)Galactosiltransferase α

( α αα ααGT) Knockout Mouse Model

Gabriela R Rossi,1 Robert C Unfer,1 Charles J Link.1

1 Tumor Immunology Section, Stoddard Cancer Research Institute,

Des Moines, IA, United States.

The glycosylation discordance due to the absence of αGalactosyl

antibodies (Ab) produced by humans against this epitope is the

major barrier for xenotransplantation The rapid rejection of cells

hyperacute response, is characterized by the binding of these Ab to

αgal epitopes and complement mediated cell destruction Based on

this concept, we have proposed that human cancer cells genetically

engineered to express these xenoantigens could represent a new

vaccination approach to treat cancer patients To test the hypothesis

that the expression of αgal epitopes in cancer cells may lead to their

was used These mice, like humans, lack αgal epitopes and can be

stimulated to produce anti-αGal Ab The murine melanoma cell line

cells were used as negative controls All 11 mice challenged

subcutaneously (SC) with native B16 cells died, as well as 11 out of

12 mice challenged with mock-transduced cells While nearly 50%

challenged for more than 80 days (9 out of 19 tumor free animals)

melanoma cells survived a second re-challenged with native B16

cells indicating that tumor immunity was induced against the αGal

negative tumor When mice were challenged intravenously (IV), a

significant reduction of lung melanoma metastasis was observed in

with mock-transduced B16 (25 vs 80, p<0.0005) Moreover, 4 out

of 11 mice that received αGal.B16 cells were free of lung metastasis

None of the 7 mice receiving B16.Mock cells were free of lung

tumors and one mouse died of pulmonary congestion We postulated

that the vaccination with whole cancer vaccine cells expressing αGal

epitopes would induce tumor immunity for the treatment of

pre-established tumors To test this hypothesis, whole cancer vaccine

cells were generated by irradiation after transduction and injected

SC to mice with pre-established SC tumors Control mice were

non-vaccinated and non-vaccinated with B16.Mock As expected 10 out of

11 non-vaccinated mice and 21 out of 24 mock vaccinated mice

developed SC melanoma tumors However, 9 out of 24 animals

receivingαGal expressing vaccines remained tumor free for at least

21 days after the challenge indicating partial success in the treatment

of this highly aggressive SC tumor model These results demonstratethat genetically engineered αGal expressing vaccine cells induceprotective immunity against the native αGal negative B16 melanomatumor increasing the long-term survival of vaccinated mice Inaddition, we demonstrated partial treatment of SC melanoma tumors

pre-clinical data supporting the concept of inducing a hyperacuterejection of xenoantigens to generate a novel cancer vaccine for thetreatment of humans with malignancies

Dr Link is also member board of NewLink Genetics

299 Syn3 Potentiates rAd-IFN Gene Therapy for Superficial Bladder Cancer

Robert J Connor,1 Heidrun Engler,1 Jennifer M Philopena,1 BillDemers,1 Duane E Johnson,1 Daniel C Maneval,1 Julia A.Jorgensen,2 Shu F Wen,2 Erlinda Quijano,2 Colin P Dinney,3

Superficial bladder cancer is an attractive target for localized genetherapy However, initial efforts to transduce the bladder usingadenoviral vectors have been unsuccessful, presumably due to thepresence of an antiadherence barrier that protects against infections

as well as the toxic effects of urine We identified an agent Syn3 thatdramatically enhances adenoviral transduction of the urothelium.Intravesical administration of adenoviral vectors in a Syn3formulation can increase transgene expression to both normalurothelium and superficial tumors For treatment of superficialbladder cancer, we believe that intravesical administration of anadenoviral vector containing a secreted gene product may be anadvantage compared to delivery of vectors encoding nuclear orcytoplasmic proteins that are confined to the transduced cell Thetherapeutic protein can be secreted into the urine from bothtransduced normal urothelium and tumor tissue, resulting in anaccumulation and potentially high local concentration of thetherapeutic protein Interferon gene therapy has shown efficacy inmouse subcutaneous tumor models Therefore, we have utilized anadenovirus encoding the human interferon a gene for our studies toevaluate the levels of interferon obtained after intravesicaladministration Delivery of rAd-IFN (7.4 x 1010 P/ml) in a Syn3formulation (1 mg/ml) results in a 10,000-fold increase in the levels

of rAd-IFN mRNA expression in mouse urothelium compared torAd-IFN without the Syn3 enhancing agent We have also comparedthe levels of interferon protein in the urine of rats administered rAd-IFN (7.4 x 1010 P/ml) either in a Syn3 or vehicle formulation Animalsthat received rAd-IFN in a vehicle formulation had no detectableinterferon in the urine In contrast, significant levels of interferonprotein (up to 125 ng/ml) were detected in the urine of rats thatreceived rAd-IFN in a Syn3 formulation Expression of interferonwas confined to the bladder, with minimal interferon detectedsystemically We evaluated the efficacy of rAd-IFN / Syn3 using anorthotopic tumor model for superficial bladder cancer using the

human TCC cell line UMUC-3 In vitro studies have shown that

rAd-IFN inhibits UMUC-3 cell proliferation UMUC-3 tumor cellswere instilled in the bladders of nude mice and tumors were allowed

to grow for one week Mice then received intravesical administration

of either rAd-IFN or a control vector in a Syn3 formulation on twoconsecutive days Animals were sacrificed 21d after tumor cellimplantation, their bladders were removed and the tumor burdenscored both macroscopically and microscopically The percentage

of tumor free animals was significantly greater in the mice thatreceived rAd-IFN in the Syn3 formulation (12/14) compared to

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mice that received a control adenovirus (2/15) Based upon our

results, we believe that rAd-IFN/Syn3 may constitute a new highly

effective treatment for bladder cancer

Drs Benedict and Dinney are employees of MD Anderson Cancer

Center

300 Antimetastatic Effects of IL-12

Gene-Modified Bone Marrow Cells in a Mouse Model of

Metastatic Prostate Cancer

Hongyu Wang,1 Guang Yang,1 Takefumi Satoh,1 Nobuyuki

Kusaka,1 Xiaorong Ji,1 Terry L Timme,1 Tetsuo Fujita,1 Taoyan

Men,1 Timothy C Thompson.1,2,3

1 Scott Department of Urology; 2 Department of Molecular and

Cellular Biology; 3 Department of Radiology, Baylor College of

Medicine, Houston, TX.

Recombinant interleukin-12 (IL-12) is a potent

immunomodulatory cytokine that has been shown to exert strong

antitumor and antimetastatic effects in various mouse models Based

on our previous studies demonstrating that IL-12 gene therapy has

specific effects against bone metastases, we used a retroviral

vector-mediated gene-modified cell therapy approach to overexpress IL-12

in adult hematopoietic stem cells to target and achieve long term

transgene expression in bone marrow and potentially other sites

where metastases develop 129/SvRosa mice were treated with

5-fluorouracil six days before isolating bone marrow cells Red blood

cells were removed from the bone marrow by ficoll-paque™ plus

centrifugation After pre-stimulation with 20 ng/ml IL-3, 50 ng/ml

IL-6, 100 ng/ml stem cell factor, 20 ng/ml granulocyte-colony

stimulating factor (G-CSF) and 50 ng/ml Flt-3 Ligand for 24 hours,

bone marrow cells were cultured in 6-well plate coated with

retronectin (25 μg/well) The murine IL-12 transducing retroviral

vector, DFG-mIL-12, or control vector, DFG-eGFP, was used for

bone marrow cell infection Bone marrow cells were infected once

daily for three consecutive days with high titer (>10 6 IU/ml)

supernatant Transduction efficiency of mouse bone marrow cells

(20-30%) was determined by intracellular murine IL-12 expression

or eGFP expression DFG-mIL-12 or DFG-eGFP transduced bone

marrow cells (106/mouse) were injected via tail vein into recipient

129/Sv mice that harbored metastases previously established by i.v

injection of 178-2 BMA mouse prostate cancer cells three days

prior to treatment The animals were sacrificed at various time points

and sera and specific tissues were collected and analyzed Sera

obtained from DFG-mIL-12 bone marrow treated mice showed a

gradual increase in IL-12 (p40, ELISA) that reached a peak (0.89 ng/

ml) at day 9 In contrast, IL-12 levels in the serum of DFG-eGFP

bone marrow treated mice were significantly lower (0.11 ng/ml)

throughout the time course Flow cytometric analysis indicated that

21days following the bone marrow cell injection, approximately

15% of peripheral blood cells stained lac-Z positive Mice treated

with DFG-mIL-12 transduced bone marrow cells had significantly

fewer metastatic lung colonies (mean=39) compared to mice treated

with DFG-eGFP transduced bone marrow cells (mean=76,

P=0.0127) or with HBSS (mean=67, P=0.0155) Histochemical

analyses showed that 80% of the mice in the DFG-eGFP bone

marrow treated group and 83% of the mice in the HBSS treated

group had bone metastases HBSS treated mice showed large

metastatic tumor deposits that extended into the connective tissues

surrounding the bone In marked contrast, only 17% of the mice had

bone metastases in the group treated with DFG-mIL-12 transduced

bone marrow cells Therefore, systemically delivered bone marrow

cells genetically engineered to produce IL-12 are effective against

pre-established metastases in this model system of prostate cancer

metastases

301 Therapy of Human Prostate and Breast Carcinoma in Experimental Models by Redirected Effector Lymphocytes Expressing Erb-B2 Specific Chimeric Receptors

Jehonathan H Pinthus,1 Dinorah Friedmann-Morvinski,1 VictoriaMelina,1 Tova Waks,1 Zelig Eshhar.1

1 Immunology, The Weizmann Institute of Science, Rehovot, Israel.

Major problems that impede the application of active vaccinationfor cancer immunotherapy are that tumor cells often escape theimmune system and do not express rejection antigens and that thepatients’ lymphocytes are tolerant and often anergic to these antigens

In order to overcome these difficulties, we took advantage of theavailability of monoclonal antibodies specific to tumor - associatedantigens and the efficiency of tumor elimination by T cells andcombined them together in the “T-body” approach The T-bodyapproach has been developed in our laboratory to expand therecognition spectrum of effector lymphocytes and redirect them topredefined targets, using chimeric receptor (CR) genes with antibody-type specificity The modular structure of the CR containing definedecto-, spacer, transmembrane and cytoplasmic domains enabled itsengineering to fit a desired task Several designs have been constructedemploying the antibody V region in the form of a scFv linked totriggering subunits of the Fc receptor (FcR) or TCR/CD3 complexes

We nicknamed T cells expressing such CR T-bodies, and havedemonstrated that they can undergo stimulation in an MHC-independent manner, yet restricted by the antibody specificity Totarget human breast tumors we have constructed several scFv’sfrom antibodies recognizing the ErbB-2 growth factor receptor that

is over-expressed on human cancer cells and in several casescontributes to the state of maligmnancy To achieve full T cellactivity against target cells that escape the immune surveillance, wecombined both antigen receptor signal with the co-stimulatory signalrequired for full T cell activation by inserting the cytoplasmic,transmembrane and part of the extra cellular regions of the CD28molecule in-between the scFv recognition unit and the gamma orzeta signaling domains For efficient retrovirally-mediatedtransduction of human PBL T cells, optimized protocols wereadopted that yields 40-80% of T cells positive for surface chimericreceptor Following introduction of such tripartite chimeric receptorconstruct into murine and human effector lymphocytes the chimericgenes have been expressed as functional receptors and conferrednon-MHC restricted, anti-erb-B2 antibody specificity on therecipient cells Specific killing and elimination of tumor cells havebeen demonstrated both in vitro and in vivo models Notable, naive

T cells derived from transgenic mice expressing the CD28 tripartite

CR underwent full stimulation by plastic-immobilized antigens andwere more resistant than wild-type cells to activation-inducedapoptosis In the SCID mouse system we have demonstrated theability of genetically engineered human T bodies to retard the growth

of, and even reject, breast and prostate cancer xenografts in significantnumber of mice Migration of the genetically programmedlymphocytes to bone metastases following systemic administrationhas been dramatically improved using several methods that increaselocal SDF-1production Such specific homing was dependent on theexpression of CXCR4 on the surface of the T-bodies These preclinicaldata should enhance the clinical application of the T-body approach

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302 Semliki Forest Virus Mediated

Intratumoral Expression of IL 12 Induces Tumor

Regression in a Mouse Model of Colon

Adenocarcinoma

Juan Roberto Rodriguez-Madoz,1 Ignacio Melero,1 Nerea

Razquin,1 Jesus Prieto,1 Cristian Smerdou.1

1 Medicina Interna, University of Navarra, Pamplona, Navarra,

Spain.

Two Semliki Forest Virus (SFV) based vectors expressing murine

interleukin-12 (IL-12) have been developed and their antitumoral

efficacy has been tested in a mouse model of colon carcinoma In one

of the vectors (SFV IL-12), genes coding for IL-12 p35 and p40

subunits were cloned under a single viral subgenomic promoter using

an IRES between them A second vector (SFV enhIL-12) carries

each gene fused to the SFV capsid translation enhancer, under

independent viral subgenomic promoters Recombinant SFV viral

particles from both vectors were produced and IL-12 expression

was tested in supernatants of BHK infected cells by ELISA IL-12

was expressed at 9 μg/106 cells from SFV IL-12 vectors and at 85

μg/106 cells from SFV enhIL-12, indicating a correct function of the

translation enhancer In both cases the protein was biologically active

as shown by the induction of IFNγ in murine splenocytes incubated

with supernatants from infected BHK cells Single tumor nodules

were implanted in the flank of C57BL/6 mice by subcutaneous

injection of MC38 colon carcinoma cells These nodules were treated

with a single intratumoral injection of SFV IL-12, SFV enhIL-12,

and SFV LacZ or saline as controls Injection of vectors encoding

IL-12 resulted in both cases in a significant inhibition of tumor

growth in a dose-dependent manner With a dose of 108 viral particles

more than 80% of treated mice experienced a complete tumor

regression with long-term tumor-free survival However, when lower

doses of vector were used SFV enhIL-12 was more efficient than

SFV 12 in inducing antitumoral responses, indicating that the

IL-12 amount expressed in the tumor is important for its therapeutic

activity In addition, all mice that rejected the tumors showed a

specific protection against tumor rechallenge CTL assays and

dependent In animals treated with IL-12 expressing vectors high

cytokine levels were found in the tumors at 24 h postinjection (2,5

ng IL-12/mg protein for SFV IL-12 and 14,4 ng IL-12/mg protein for

SFV enhIL-12) but decreasead along time until day 6 A similar

result was found in serum although the cytokine levels were much

lower These data show that alphavirus vectors can be useful to

enhance antitumor immunity by local delivery of functional

cytokines, such as IL-12

303 Drug Resistant Immunotherapy: Strategies

Combining Chemotherapy and Immunotherapy

Douglas W McMillin,1 David Kotzbauer,1 Becker Hewes,1 H

Trent Spencer.1

1 Pediatrics, Division of Hematology/Oncology, BMT, Emory

University School of Medicine, Atlanta, GA.

New treatment options such as allogeneic bone marrow

transplantation and immunotherapy have provided hope for patients

suffering from various types of cancer, but treatment of late-stage

or metastatic cancer is still extremely problematic Treatments using

tumor vaccines are emerging for these patients that may greatly

improve their outcomes, but it is predicted that immunotherapy

alone may not be sufficient to completely eradicate tumor growth

Combining chemotherapy agents with tumor vaccines has proven

difficult because vaccines are not likely to stimulate significant

immune responses in a chemotherapy-induced immunosuppressive

setting Therefore, we are currently investigating strategies that will

allow both approaches to be used, through the development of

tumor cell vaccines and the generation of genetically engineered drugresistant T-lymphocytes, a process we call drug resistantimmunotherapy Our approach is to generate immunogenic geneticallyengineered tumor cells ex vivo by transient expression of cDNAsencoding immunostimulatory molecules (anti-4-1BB, GM-CSF, IL-

12, and MIP-3alpha) Our optimized transfection protocol usingLipofectamine reagents routinely achieves >80% transfectionefficiency of sarcoma, neuroblastoma, or carcinoma mouse cell lines.Based on the specific genes introduced into the tumor cells, weshow that transient expression is sufficient to induce dendritic cell

or T-cell activation, and that the activated cells mount an effectiveanti-tumor immune response that can eradicate the growth of mousetumor models such as Ag104 Spleenocytes are then harvested fromvaccinated mice and transduced with retroviral vectors encodinggenes that confer drug resistance We have developed novel MSCV-based vectors that confer high-level resistance against the anti-neoplastic agents methotrexate, trimetrexate, raltitrexed, 2-CdA, andcamptothecin Using ecotropic virus on fibronectin coated plates

we achieve >50% transduction of tumor specific T-cells, as measured

by tetramer staining, which retain their cytotoxic activity ex-vivo,and based on our studies showing in vivo protection of hematopoieticprogenitor cells, we anticipate gene modified T-cells will remaindrug resistant in vivo Because the immunocompetent cells areengineered to withstand drug challenges it will be possible toadminister chemotherapy and immunotherapy concurrently, andhopefully increase the effectiveness of anti-cancer cellular therapies

304 Immunogenetherapy Improves Outcome and Prolongs Survival in Early and Advanced Non-Small Cell Lung Cancer

R J Kruklitis,1 S Singhal,1 J Greenberg,1 V Kapoor,1 P

DeLong,1 D Sterman,1 L R Kaiser,1 S M Albelda.1

1 Thoracic Oncology Research Lab, University of Pennsylvania, Philadelphia, PA.

INTRODUCTION: Viral gene transfer of Ad.IFN-β and

Ad.IL-12 is effective at stimulating an anti-tumor immune response againstseveral malignancies including malignant mesothelioma Our firstgoal was to translate this knowledge into management of murinemodels of non-small cell lung cancer (NSCLC) Our second goal was

to improve outcome in large tumors (>800 mm3) by combiningimmunotherapy with surgical debulking

MATERIALS/METHODS: BALB/c or C57/Bl6 mice underwent

subcutaneous injection with several syngeneic NSCLC (L1C2, LLC1,

control Ad.LacZ (n=55) virus was used to treat mice with varioussize flank tumors (100-1000 mm³) For larger tumors, surgicaldebulking was used to decrease the tumor burden afterimmunogenetherapy Animals were assessed for rate of tumorgrowth, time to recurrence and overall survival Growth of distantmetastatic foci was also monitored Tumor infiltration by Tlymphocytes was quantified using immunohistochemistry.Splenocytes or purified CD8+ T lymphocytes from mice treatedwith immunogenetherapy were simultaneously injected with tumorcells into naive mice (Winn assay)

RESULTS: In mice with various small (<200 mm³) NSCLC

tumors, treatment with either Ad.INFb or Ad.IL-12 versus Ad.LacZwas effective at preventing tumor progression (85% versus 12%,p<0.01), and in some instances, causing tumor eradication (22% oftreated mice) For mice with larger tumor burdens (>800 mm³), viralgene transfer did not prevent tumor growth Although anti-tumorCD8+ cytotoxic T lymphocytes (CTLs) were present followingtreatment with Ad.INFb and Ad.IL-12 versus Ad.LacZ (87% vs 12

%, p<0.01), the CTLs failed to infiltrate these tumors The addition

Ad.LacZ, resulted in a delay of local tumor recurrence (mean

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recurrence time: 27 days vs 11 days, p<0.01), an increase in overall

survival (80% vs 20%, p<0.01), and a 10-fold smaller size of

implanted tumor cells (42 mm³ vs 480 mm³, p<0.01, at 10 days)

CONCLUSION: These data demonstrate that for several NSCLC

cell lines, Ad.IL-12 and to a lesser extent, Ad.IFN-β prevent tumor

growth and prolong survival For large tumors, combining Ad.IL-12

with surgical debulking significantly reduced tumor recurrence and

prolonged survival This benefit resulted from Ad.IL-12 stimulating

an effective antitumor immune response; Ad.IL-12 increased the

number of CD8+ T lymphocytes and enhanced their ability to

infiltrate small tumor nodules Preoperative immunogenetherapy

offers a novel strategy to improve survival in patients with surgically

resectable disease and to qualify unresectable patients for surgical

intervention

305 Anti-Melanoma Immune Responses

Induced by Autologous Tumor Cell Vaccine

Engineered To Express Interleukin (IL)-12 and

IL-18 by Means of the EBV/lipoplex

Hidetsugu Asada,1 Tsunao Kishida,1 Hideyo Hirai,1 Yoichi

Iwakura,2 Jiro Imanishi,1 Osam Mazda.1

1 Microbiology, Kyoto Prefectural University of Medicine, Kyoto,

Kyoto, Japan; 2 Center for Experimental Medicine, Medical

Science, University of Tokyo, Minato-ku, Tokyo, Japan.

Genetically manipulated tumor vaccines potentially induce

anti-tumor immune responses, resulting in significant therapeutic

outcome against primary as well as metastatic tumors We have

previously shown the effectiveness against B16 melanoma of

autologous tumor cell vaccine that had been engineered to express

IL-12 and IL-18 using the Epstein-Barr virus (EBV)-based plasmid

vector/cationic liposome system (the EBV/lipoplex) Thanks to the

highly efficient transfection and expression capabilities of the EBV/

lipoplex, a large proportion of tumor cells strongly express the

cytokines several days after the transfection, so that the cells could

be used as vaccines without subsequent drug selection or cloning In

the present study immune responses elicited in vivo by the vaccine

were also investigated

Materials and Methods

repetitive injections of GK1.5, 2.43, and anti-asialo GM1 antibodies,

cells (day 0) followed by vaccination on days 5, 12, 19 and 26 with

irradiated B16 cells that had been transfected by cationic lipid with

EBV-plasmid vectors encoding IL-12 and/or IL-18 (B16/mIL-12,

B16/mIL-18, and B16/mIL-12+mIL-18) CTL and NK activities

were evaluated by standard 51Cr release assay Pulmonary metastasis

cells via the tail vein (day 0), followed by vaccinations on days 1, 6,

and 11

Results

The B16/mIL-12 vaccination effectively inhibited the growth of

the tumors pre-established in normal mice, which survived

significantly longer than controls In contrast, IL-18 alone failed to

show significant therapeutic outcome Strong cytotoxic activity

against B16 cells was demonstrated in the spleen cells from

tumor-bearing mice that had been immunized with B16/mIL-12 or B16/

mIL-12+mIL-18 Vaccines producing IL-12, IL-18 or both augmented

were implanted with B16, subsequent B16/mIL-12 immunization

did not result in any therapeutic outcome The B16/mIL-12 vaccine

showed reduced, but still significant, anti-tumor effects in the mice

lacking CD8+ T cells, while CD4+ T cell deprivation completely

abrogated the vaccine effect In contrast, depletion of NK cells did

not affect the therapeutic results The 12 and 12+mIL-18 also successfully suppressed pulmonary metastasis ofmelanoma that had been intravenously challenged

B16/mIL-DiscussionThe IL-12 produced from the vaccine successfully elevated CTLand NK activities IFN-γ and CD4+ T cells are a prerequisite to theanti-tumor immunity, while NK cells are not necessarily required.CD8+ T cells may partially contribute to the anti-tumor effects Thepresent study strongly suggests that the EBV/lipoplex is quite useful

in manipulating tumor vaccine that is effective without drug selection

or cloning, and IL-12 functions as powerful adjuvant triggering Th1response against solid and metastatic melanomas

306 Stent-Mediated Gene Transfer: A Novel and Efficient Means of Gene Delivery to the Tracheobroncial Tree

R J Kruklitis,1 I Fishbein,2 S Singhal,1 J Greenberg,1 V

Kapoor,1 S M Albelda,1 R Levy,2 D Sterman.1

1 Thoracic Oncology Research Lab, University of Pennsylvania Medical Center, Philadelphia, PA; 2 Children’s Hospital of Philadelphia, Philadelphia, PA.

Rationale: Gene therapy has been proposed as a means of treating

a variety of airway diseases Unfortunately, gene transfer to humanbronchial epithelium has proven to be complicated and inefficient

The goal of this study was to assess in vitro and in vivo

stent-mediated gene delivery to human bronchial epithelial cells and small cell lung cancer (NSCLC) cells

non-Methods:Replication-incompetent adenoviral vectors (Ad5,

E1-/E3-), containing either reporter genes (Ad.lacZ, Ad.GFP) or

therapeutic genes (Ad.INF-β) were tethered to coronary stents orcircular mesh disks made of stainless steel These appliances werethen assayed for their ability to transfect both murine and humanNSCLC cells (L1C2, A549) and bronchial epithelial cells Genetransfer was confirmed using scanning fluorescent microscopy forAd.GFP, and beta-galactosidase (β-gal) staining for Ad.lacZ β-gal

staining was performed on either sectioned tissues or on wholetissues after formaldehyde fixation

Results: We demonstrated that murine and human NSCLC cells

could be successfully transfected with reporter genes in vitro Greater

than 40% of A549 cells were transfected with Ad.GFP-complexedmesh compared with less than 2% of cells with an equivalent amount

of soluble virus (p<0.01) Ad.lacZ-complexed mesh also successfully

transfected marker gene into established murine flank NSCLC

significantly slowed the growth of flank NSCLC tumors compared

with control Ad.lacZ virus (291 mm³ vs 636 mm³, p<0.05) In

addition we demonstrated that stents complexed with reporter genescould successfully transfect intact rat and human bronchialepithelium Specifically, we achieved efficient stent-mediated gene

transfer with Ad.GFP and Ad.lacZ to explanted human bronchus in

an ex vivo culture system, and with Ad.lacZ to rat tracheal epithelium

in situ

Conclusions: Stent-mediated gene transfer provides a highly

efficient means of delivering both marker and therapeutic genes totarget cells in rat and human airways The superior transfectionefficiency may provide a means of overcoming the difficultiespreviously encountered, in particular by lowering requisite vectortiters This technology may prove useful for treating a variety ofcongenital and acquired tracheobronchial diseases including NSCLC,cystic fibrosis and asthma

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307 Intravesical Gene Therapy for Superficial

Bladder Cancer with Syn3/Ad- α αα αα IFN

Ziming Tao,1 Robert J Connor,2 Chang-Soo Kim,1 Jain-Hua

Zhou,1 Xinqiao Zhang,1 Heidrun Engler,2 Daniel C Maneval,2

William Demers,2 Colin P N Dinney,3 William F Benedict.1

1 Genitourinary Medical Oncology, UT MD Anderson Cancer

Center, Houston, TX, United States; 2 Canji Incorporated, San

Diego, CA, United States; 3 Urology, UT MD Anderson Cancer

Center, Houston, TX, United States.

The efficacy of intravesical Syn3/Ad-α IFN gene therapy using a

superficial bladder cancer tumor model we have recently developed

has been evaluated Syn3 was used to increase adenoviral-mediated

gene transfer In this model, we are able to monitor the progression

of human bladder cancer cells containing the green fluorescent protein

(GFP) after they have produced superficial tumors in the bladders

of athymic mice Prior to intravesical treatment, the tumor burden in

the bladder of each mouse was quantitatively imaged Each mouse

then received subsequent intravesical administration of either 100μl

x 1011 P/ml; Syn3: 1 mg/ml) for 1 hour on two consecutive days

Three weeks post treatment, the bladders were re-exposed and

imaged for changes in tumor size The mice were then sacrificed

Their bladders were instilled with formalin and embedding in paraffin

for histological evaluation When tumor burden was compared before

and after treatment, only bladders that received Ad-α IFN in a Syn3

formulation had significant decrease in tumor size, whereas bladder

Syn3 alone treated mice (p< 0.0001) Histological analysis of multiple

sections from each bladder confirmed that the tumor burden in the

Syn3/Ad-IFN treated animals was marked reduced, corresponding

to the imaging results with minimal local toxicity to normal urothelium

being observed These results suggest that intravesical

Syn3/Ad-IFN therapy may be a new treatment paradigm for superficial bladder

cancer

Drs Connor,Engler,Maneval and Demers are employees of Canji/

Schering Plough who provided the Syn3 and AdINF

308 Fractionated Radiation Alone Protects

C57BL Mice from Challenge with CMT Cells If

Challenged within 28 Days from the Completion

of Radiation

Kenneth R Olivier,1 Marka R Crittenden,2 Michael J Gough,2

Richard G Vile,2 Kevin J Harrington.3

1 Radiation Oncology, Mayo Clinic, Rochester, MN, United States;

2 Molecular Medicine, Mayo Clinic, Rochester, MN, United States;

3 Institute of Cancer Research, Chester Beatty Laboratories,

London, United Kingdom.

Vaccination of cancer patients will likely occur within the context

of conventional therapy Therefore we investigated the effect of

fractionated radiation therapy on the vaccination of C57BL mice

against the murine colorectal cancer line CMT Vaccination consisted

of the subcutaneous (SQ) administration of irradiated CMT cells

given twice, seven days apart A challenge of CMT cells was given

SQ in the contralateral flank 35 days after final vaccination Radiation

consisted of 40 Gy of 300 KVp photons delivered in 15 fractions on

consecutive days to the lower hemibody of anesthetized mice The

vaccinations were given before, during or after the course of radiation

These three groups were compared to radiation alone with challenge,

vaccination alone, and challenge alone The results show the control

group of vaccination alone protected 100% of the mice The control

group of challenge alone resulted in the sacrifice of 70% of the mice

for tumor There was no significant difference in vaccination efficacy

between the before, during or after radiation groups as compared

with vaccination alone Surprisingly, there was also no difference

between radiation alone with challenge at 14 or 28 days aftercompleting radiation compared with vaccination alone with 85%and 90% of mice, respectively, surviving tumor free Radiation alonewith challenge at 42 days was significantly worse (p=0.0001) thanthe other radiation alone timepoints, and not significantly differentthan challenge alone with 100% of the mice sacrificed for tumor Weconclude that radiation did not interfere with the vaccination ofC57BL mice regardless of whether the radiation was given before,during or after radiation A novel and intriguing finding is theprotection conferred by radiation alone if the challenge occurredwithin 28 days of completing radiation It remains to be seen if thissurprising protection is a result of the local or systemic effects ofthe radiation Experiments are on going to further elucidate thiseffect

309 Characterization of a Tumor Model Derived from a C57/BL6

α αα

αα(1,3)Galactosyltransferase Knock-Out Mouse

Daniel J Hellrung,1,2 Charles J Link.1

1 Gene Therapy, Stoddard Cancer Research Institute, Des Moines, IA; 2 Immunobiology, Iowa State University, Ames, IA.

Alpha (1,3)galactosyltransferase is the major xenoantigenassociated with hyperacute rejection of xenotransplants Tumorvaccines engineered to express this gene may show promise inbreaking tumor tolerance However there has been a lack of alternativetumor models to the highly published B16 with which to study theimmunology associated with α(1,3) galactotyltransferase modified

tumor vaccines Moreover, limited in vivo models exist to study the

basic biology of α(1,3) galactotyltransferase in xenotransplantationexperiments Therefore, we have developed and characterized anadenocarcinoma of the small intestine, CA320M, derived from C57/BL6α(1,3)galactosyltransferase knock-out mice Mice were subject

to two intraperitoneal injections of various concentrations of dimethyl-1,2-benz-anthracene and 3-methylcholanthrene After a4-month latency period animals began presenting with tumors.CA320M was harvested from a female mouse, analyzed byhistopathology and established in culture CA320M failed to bind

9,10-Griffonia simplicifolia IB4 FITC labeled isolectin, specific for theαgal epitope, as compared to cells engineered to express the murineα(1,3)GT gene Flow cytometric analysis demonstrated a normaldiploid genome To determine the putative tumoragenic potential ofthis tumor line, it was subjected to serum starvation conditions tosimulate the microenvironment upon initial subcutaneous injection.CA320M cultured in 0.1% fetal bovine serum (FBS) for 48 hrsdemonstrated no appreciable difference in cell cycle as compared to

cells cultured in 10% FBS In vivo tumor growth curves in syngeneic

CA320M cells injected subcutaneously resulted in 200mm³ tumorswithin 14 days which increased to 500mm³ by day 30

Histopathology of transplanted tumors after 14 days growth in vivo demonstrated a morphology consistant with fibrous connective

tissue The addition of an α(1,3)galactosyltransferase knock-outtumor model like CA320M will allow investigators to further explorethe use of αgal mediated tumor vaccines and enhance the study ofxenotransplantation in a small animal model

Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts S123

310 Pancreatic Cancer Escape Variants That

Evade Immuno-Gene Therapy through Loss of

Sensitivity to IFN- γγγγγ Induced Apoptosis

Guillermo Mazzolini, Iñigo Narvaiza, Alfonso Martinez-Cruz,

Ainhoa Arina, Miguel Barajas, Juan Carlos Galofre, Cheng Qian,

Jose Maria Mato, Jesus Prieto, Ignacio Melero

1 Gene Therapy Unit FIMA, University of Navarra, Pamplona,

Navarra, Spain.

Combined injections into experimental tumor nodules of

adenovirus encoding IL-12 and certain chemokines are capable to

induce immune-mediated complete regressions In this study we

found that the combination of two adenoviruses, one encoding

successful in treating CT-26 derived colon carcinomas However, in

experimental tumors generated from the pancreatic carcinoma cell

line Panc02 such combined treatment induces 50% of macroscopic

complete regressions, although local relapses within one week are

almost constant We derived cell lines from such relapsing tumors

and found that experimental malignancies derived from their inoculum

were not amenable to treatment in any case with

insensitive to in vitro induction of apoptosis by IFNγ, in clear

contrast with the original Panc02 cells Comparative analyses by

cDNA arrays of relapsing cell lines versus wild type Panc02 were

performed revealing an important number of genes (383) whose

expression levels were modified more than two-fold These changes

grouped in certain gene ontology categories and should harbor the

mechanistic explanations of the acquired selective resistance to IFNγ

Interestingly, the expression at the RNA level of the

apoptosis-related protein clusterin is completely lost in the studied IFN

γ-resistant cell variants

311 An Evaluation of the Use of IL-1H4

Adenovirus To Induce Protein Production and

Reduce Tumor Burden

Mary Donahee,1 Karen Kozarsky,1 Deborah Welham.1

1 Protein Agents and Human Gene Therapy, GlaxoSmithKline,

King of Prussia, PA.

IL-1H4/IL-1F7 is a 22 kD cytokine in the Interleukin(IL)-1 family

that shares significant sequence homology with IL-18 Like IL-18,

IL-1H4 lacks a characteristic leader peptide but contains a propeptide

domain that is cleavable by members of the caspase family IL-18

has significant antitumor effects in mouse models; therefore,

IL-1H4 was evaluated as an antitumor agent Adenovirus vectors

encoding IL-1H4 and IL-18 were used to obtain sustained protein

expression in mice injected with a mouse sarcoma line Prior to in

vivo testing, verification of protein expression was performed in

vitro Lysates from A549, Huh-7 and HeLa cells that were transduced

with Ad.IL-1H4 showed both the 22 kD mature form of the IL-1H4

protein as well as the 26 kD pro form while only low levels of

secreted IL-1H4 were detected in the media In vivo, MCA205

tumor cells were introduced into C57Bl/6 male mice 2 days prior to

treatment with 1 x 10 11 viral particles of adenovirus The treatment

groups receiving the Ad.IL-1H4 or Ad.IL-18 showed a 5 day delay

in tumor growth over the control groups Subsequent experimentation

was performed using a dual infection with both Ad.IL-1H4 and

Ad.IL-18 Again a trend toward tumor growth reduction was seen

The group receiving Ad.IL-1H4 showed a 5 day delay in tumor

growth vs the control group while the Ad.IL-18 showed a 7 day

delay The group receiving both Ad.IL-1H4 and Ad.IL-18 also

showed a 7 day delay in tumor growth, indicating no obvious

synergistic effect when combining the viruses

312 Phase I Trial of Repeated Intralesional Injection of TG1024 (Adenovirus-Interleukin-2) in Melanoma and Accessible Solid Tumours

Rochlitz Christoph,1 Morcinek Jessica,3 Reuter Juergen,1 SlosPhilippe,2 Squiban Patrick,2 Dummer Reinhard.3

1 Medical Oncology, Kantonsspital, Basel, Switzerland;

2 Transgene, Strasbourg, France; 3 Dermatology, University Hospital, Zurich, Switzerland.

Many solid tumors cannot be completely removed by surgeryand do not respond to radio or chemotherapy However, they areaccessible to direct injection of agents such as gene therapy vectors

A percentage of some advanced solid tumors, melanoma and renalcell cancer respond to systemic Interleukin-2 (IL-2) and/or interferon.However, these are associated with significant toxicities Localproduction of IL-2, by direct injection of a gene transfer agent,should result in local production of IL2, and associated localbiological impact, but with lower systemic toxicities The TG1024product is made of a suspension of non-replicating (E1 and E3regions deleted) recombinant adenoviral particles containing a humanIL-2 cDNA insert We undertook a phase I, open-label, dose-escalating trial of repeated intratumoral administration of TG1024

in patients with advanced melanoma and other accessible solidtumors Twenty patients (12 melanoma and 9 other solid tumorswere enrolled in 5 successive cohorts at the following TG1024 doses:3x10E8 total particles (tp), 3x10E9 tp, 3x10E10 tp, 8x10E10 tp and3x10E11 tp Patients received intratumoral injections of TG1024into the designated lesions on days 1, 21, and 42 (1 treatment cycle)and thereafter up to 4 cycles, if there was no evidence of progressivedisease (PD) Blood samples were taken at baseline, before injection,

6 hours after injection and 1 week after injection and cytokine levelsevaluated A dose dependent production of circulating IL2 was noted.IL2 production was particularly striking in melanoma patients.Repeated administration was associated with repeated cycles ofdetectable IL2 in the circulation of several patients Treatment waswell tolerated Most common adverse event were injections sitereactions Only 2 grade III (fever, transient lymphopenia) adverseevents were noted and 3x10E11 tp has been considered as themaximum tolerated dose Evaluations of the patients clinicalresponses are still are ongoing and will be presented Our resultsshow that local administration of an IL2 gene transfer agent canresult in the production of significant levels of IL2, but does notresult in the severe toxicities associated with systemic administration

of IL2 This information is of importance in the design of new genedelivery-based therapeutics in cancer

Principal Investigator and Consultant

313 Results of Clinical Study Phase 1 with Adenovirus-Interferon- γγγγγ (TG1042) in Primary

Cutaneous T and B Cell Lymphomas

Urosevic Mirjana,1 Mayer Tania,1 Morcinek Jessica,1 AcresBruce,2 Slos Philippe,2 Squiban Patrick,2 Burg Gunter,1 DummerReinhard.1

1 Dermatology, University Hospital, Zurich, Switzerland;

2 Transgene, Strasbourg, France.

During the progression of primary cutaneous lymphomas (CL),production of the Th1 cytokines decreases with a shift towards theimmunosuppressive Th2 phenotype This group of diseases hasbeen therefore successfully treated with interferons (IFNs),counterbalancing the Th2-skewing state We undertook a phase I,open-label, dose-escalating trial of repeated, intratumoraladministration of TG1042 in patients with advanced primarycutaneous T cell lymphomas (CTCL) and multilesional cutaneous

B cell lymphomas (CBCL) TG1042 product is a suspension ofnon-replicating (E1 and E3 regions deleted) recombinant adenoviral