623 inhibition of NF kb in fusogenic membrane glycoprotein causing HL 60 cell death: implications for acute myeloid leukemia

623 Inhibition of NF kB in Fusogenic Membrane Glycoprotein Causing HL 60 Cell Death Implications for Acute Myeloid Leukemia Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American[.]

Molecular Therapy Volume 17, Supplement 1, May 2009

CANCER - TARGETED GENE THERAPY II

adenoviral E2-late promoter plays an important role in the adenoviral

life cycle (Mantwill et al., Cancer Research, 2006) With this

knowledge we have developed different oncolytic adenoviral vectors

in which the transactivation domain CR3 of E1A was ablated to target

YB-1 positive tumor cells The application of these vectors causes a

strong antitumor effect in glioma cell lines which could be achieved

at lower pfu/cell, when combined with chemotherapy Additionally,

a glioma xenograft mouse model with U87 tumors was established

Animals treated with the YB-1 dependent oncolytic adenovirus

showed signifi cantly smaller tumors than untreated controls and the

effect was even enhanced by the combination with temozolomide,

leading to complete regression of two from six examined tumor

bearing mice which was verifi ed by bioluminescence imaging and

histological studies In addition, histological evaluation concerning

lymphocytic infi ltration and induction of apoptosis were used to

characterise the mode of action of the different treatments Our results

have shown the feasibility of YB-1 dependent virotherapy and form

the basis for a phase I/II clinical study

Oncolytic Adenovirs to Ex Vivo Biological Imaging

of Human Circulating Tumor Cells

Futoshi Uno,1 Yuuri Hashimoto,2 Toru Kojima,1 Shunsuke

Kagawa,1 Hiroshi Tazawa,1 Yasuo Urata,2 Noriaki Tanaka,3

Toshiyoshi Fujiwara.1

1 Center for Gene and Cell Therapy, Okayama University Hospital,

Okayama, Japan; 2 Oncolys BioPharma, Inc, Tokyo, Japan;

3 Department of Surgery, Okayama University Graduate School,

Okayama, Japan.

The presence of circulating tumor cells (CTC) in the peripheral

blood is associated with short survival and, therefore, the detection

of CTC is clinically useful as prognostic factors of disease outcome

and/or surrogate markers of treatment response Recent technical

advances including immunocytometric analysis and quantitative

real-time PCR assay have made possible to detect a few CTC in the

blood; however, there is no sensitive assay for detecting viable CTC

Here we report a new approach to visually detect live CTC among

millions of peripheral blood leukocytes using telomerase-specifi c

replication-selective adenovirus expressing green fl uorescent protein

(GFP) We constructed a GFP-expressing attenuated adenovirus, in

which the telomerase promoter regulates viral replication (OBP-401,

TelomeScan) We used OBP-401 to establish a simple ex vivo method

for detecting viable human circulating tumor cells in the peripheral

blood The detection method involves a three-step procedure including

the lysis of red blood cells, the subsequent addition of OBP-401

to the cell pellets, and the automated scan under the fl uorescent

microscope OBP-401 infection increases the signal-to-background

ratio as a tumor-specifi c probe, because the fl uorescent signal can

be amplifi ed only in tumor cells by viral replication We evaluated

23 samples obtained from histologically-confi rmed gastric cancer

patients Although the CTC level varied widely, ranging from 0 to

47 in 5-ml samples, 14 patients had more than 1 CTC In the two

cases that underwent surgery, the CTC level dropped after 4 weeks

of complete resection These results suggest that enumeration of

CTC might be useful for monitoring the effi cacy of treatment This

GFP-expressing virus-based simple method has the potential to allow

physicians to assess the response to treatment as well as disease

outcome as a relevant clinical parameter, especially in patients without

elevated levels of tumor markers

Cancer Diagnosis, Prognosis, and Therapy

Ruian Xu,1,2 Qizhao Wang,1,2 Yong Diao,1,2 William Xu,3 Habib Nagy,4 Weidong Xiao.5

1 Engineering Research Center of Molecular Medicine, Ministry

of Education, Quanzhou, Fujian, China; 2 Institute of Molecular Medicine, Huaqiao University, Quanzhou, Fujian, China;

3 Faculty of Science, University of New South Wales, Sydney, NSW, Australia; 4 Children’s Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA.

Lung cancer is the leading cause of death from cancer in the world Although the molecular network of lung carcinogenesis has been partly known at the levels of genes and proteins, and personalized therapy based on the genetic changes has made considerable progress

in the last decade, the high mortality rate is not markedly changed microRNAs (miRNAs), a class of short endogenous RNAs, acting

as post-transcriptional regulators of gene expression, are similar with siRNAs in both the biosynthesis and the function steps While, miRNAs mostly silence gene expression by binding imperfectly matched sequences in the 3’ UTR of target mRNA, which is different with siRNAs by targeting ORF of mRNA with a perfectly complementary manner miRNAs have multiple functions in lung development, and abnormal expression of miRNAs could lead to lung tumorigenesis The different expression profi les of miRNAs in lung cancer, and the stability of miRNAs in serum, all together make them

as new potentially clinical biomarkers for diagnosis and prognosis Moreover, miRNAs may serve as either novel potential targets acting

directly as oncogenes (e.g miR-17-92 cluster) or directly therapeutic molecules working as tumor suppressor genes (e.g let-7 family)

RNAi technology based on miRNAs has many advantages over

siRNAs, such as in vivo stablility, highly RNA promoter-compatiblity

and no overt toxicity Eventually, it might overcome the present disadvantages, and is used for lung cancer therapy

Membrane Glycoprotein Causing HL-60 Cell Death: Implications for Acute Myeloid Leukemia

Li Tan,1 Wenlin Huang,1 Jiangxue Wu,1 Hongyun Jia,1 Yufang Zuo.1

1 State Key Laboratory, Cancer Center, Sun Yat-sen University, Guangzhou, China.

Acute myeloid leukemia (AML) is a serious hematologic cancer with the accumulation of abnormally differentiated myeloid cells that are not mature Although, the development of better chemotherapy regimens has improved remission induction and overall survival, relapse is common and long-term survival rates remain low Resistance to standard chemotherapeutic drugs is an important cause of the relapsed, refractory leukemia to which most patients succumb, so relapse and resistance remain a signifi cant problem A unique gene therapy approach for human cancers is transduction with viral FMGs Viral fusogenic membrane glycoproteins (FMGs) are new therapeutic genes for the control of tumor growth, the cellular mechanisms mediating cell death is non-apoptotic FMG transfection

is a much more effective treatment for killing human tumor lines in vitro than commonly used suicide genes, such as melanoma tumors, glioma cell lines and hepatocellular carcinoma cell lines Here, we showed FMG expression in HL-60 cells leaded to the formation of multinucleated syncytia and cell death, the main death mode of cells

is necrosis Overexpression of HSP70 in HL-60 cells mediated by Gibbon Ape leukemia virus hyperfusogenic envelope protein (GALV-FMG) inhibited the nuclear translocation of p65, the transcriptive

activity of NF-kB and prevented the degradation of I-kB NF-kB

may negatively regulate HSP70 expression, which made a positive feed back loop for expression of HSP70 Overexpression of HSP70

Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy

S238

HEMATOLOGIC-HEMOPHILIA

in tumor cells may increase their intrinsic immunogenicity So this

form of cell death should be effective in vivo, gene therapy basing

on FMG deserve further study for the treatment of AML

Hematologic-Hemophilia

Large Animal Model for Hemophilia A Gene

Therapy

Christopher D Porada,1 Chad Sanada,1 Josh A Wood,1 Jay N

Lozier,3 Charles Long,4 Mark Westhusin,4 Duane Kraemer,4 Graça

Almeida-Porada.1

1 Anim Biotech., Univ of Nevada, Reno, NV; 2 NIH Clinical Center,

Bethesda, MD; 3 Texas A &M Univ., College Station, TX.

Hemophilia A (HA) is the most common severe hereditary

coagulation disorder, affecting 1 in 5000 males Animal models in

dog and mouse have been developed and used to study FVIII function

and to evaluate gene therapy-based treatments Unfortunately, for

unknown reasons, results obtained using these models hasn’t always

resulted in successful therapies when applied to humans Due to its

striking physiological and anatomical similarities to humans, sheep

are considered an ideal model to study a vast array of pathologies

We employed a variety of reproductive technologies to re-establish

an extinct line of sheep with a bleeding disorder that closely mimics

severe human HA These animals exhibited prolonged bleeding

from the umbilical cord that promptly stopped upon administration

of purifi ed human FVIII (hFVIII) concentrate Blood collected prior

to FVIII administration showed that these animals had almost

non-existent levels of FVIIIc and extremely prolonged PTT, with normal

levels of platelets, fi brinogen, FVII, FIX, and vWF All animals that

survived birth have developed clinical symptomatology closely

mimicking that of severe human HA, with each of these animals

exhibiting multiple episodes of severe spontaneous bleeding including

hemarthroses, muscle hematomas, and hematuria, all of which have

responded to hFVIII Thus far, inhibitors to hFVIII have been detected

in 4 treated animals, further validating the clinical relevance of this

model RT-PCR of mRNA from the spleen of normal sheep, followed

by overlapping sequence analysis allowed us to walk along the mRNA

and obtain the sequence of the complete 6765 nucleotide coding

sequence for ovine FVIII, which is translated into a 2254 amino acid

protein BLAST alignment to hFVIII protein revealed a high degree

of identity in all regions except the B domain (which is dispensable

for clotting activity in humans) Specifi cally, the A1 domain showed

81% identity, A2: 88%, A3: 87%, C1: 90%, and C2: 86%, while the B

domain exhibited only 47% identity Analysis of mRNA isolated from

the spleen of a deceased HA sheep identifi ed an 11bp region in exon 14

that differed between the wild type and the hemophiliac Importantly,

this difference introduced a premature stop codon at base position

3112-4 in exon 14, as is seen in some human patients with HA This

mutation also included a single nucleotide insertion, introducing a

frame shift which created 5 additional stop codons within the next

183bp A PCR-based RFLP analysis allows us to unequivocally

distinguish sheep that are wild-type, heterozygous, or homozygous

for the HA mutation, greatly facilitating studies using these sheep

Given the close physiologic similarity between sheep and humans,

the high degree of identity in their FVIII protein, and the decades

of experience using the sheep to study both normal physiology and

a wide array of diseases, we hope that this large animal model will

contribute to a better understanding of HA and the development of

novel gene therapy-based approaches that can directly translate to

human patients with HA

Encoding a B Domain Variant FVIII/N6 cDNA Reduced Inhibitory Anti-FVIII Antibody Titer in Hemophilia A Mice

Dominika Jirovska,1 Peiqing Ye,1 Carol H Miao.1,2

1 Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA; 2 Pediatrics, University of Washington School

of Medicine, Seattle, WA.

Due to the large size of FVIII, a B-domain deleted FVIII (BDD-FVIII) cDNA is usually used for developing gene therapy protocols for treating hemophilia A Ineffi cient transcription of wild type (WT) FVIII cDNA can be overcome by deletion of the heavily glycosylated B-domain encoding portion of the gene BDD-FVIII is as clinically effi cacious and not more immunogenic than full-length recombinant FVIII More recently, it was demonstrated that a partial deletion of the B-domain leaving an N-terminal 226 amino acid stretch containing

6 putative asparagine-linked glycosylation sites intact (FVIII/N6)

was able to increase in vitro and in vivo secretion of FVIII by 10-15

fold We have inserted this B domain variant FVIII/N6 cDNA into our liver-specifi c gene expression vector The resulting construct, FVIII/N6 plasmid was delivered into the hemophilia A mouse liver

by the hydrodynamic method In control mice treated with FVIII plasmid containing the BDD-FVIII cDNA, FVIII expression levels dropped to undetectable levels at 2 weeks post injection and high-titer anti-FVIII antibodies were generated in all the mice However,

in mice treated with FVIII/N6 plasmid, one out of fi ve mice never developed inhibitory antibodies and still had some FVIII gene expression (∼10%) at 8 weeks post gene transfer Except for one mouse which developed high-titer inhibitory antibodies, all other 3 FVIII/N6 plasmid-treated mice developed anti-FVIII antibodies with signifi cantly reduced inhibitor titer The CD4+ T cells isolated from mice injected with FVIII/N6 constructs proliferated less in response

to FVIII stimulation than those from mice injected with BDD-FVIII These results indicated that FVIII/N6 protein is less immunogenic than BDD-FVIII Interestingly, both BDD-FVIII and FVIII/N6 constructs produced similar levels of FVIII gene expression following nonviral gene transfer These fi ndings suggest that in combination with a FVIII/N6 construct, induction of long term tolerance against FVIII may be achievable with a reduced dosage and duration of the immunomodulation protocol

FVIII (cFVIII) Using Liver Directed AAV Mediated Expression of cFVIII in Hemophilia A Dogs with Inhibitor Antibodies

Jonathan D Finn,1 Denise E Sabatino,2 Margareth C Ozelo,3

ShangZhen Zhou,1 David Lillicrap,3 Timothy C Nichols,4 Valder

R Arruda.1,5

1 Children’s Hospital of Philadelphia, Philadelphia, PA; 2 University

of Pennsylvania School of Medicine, Philadelphia, PA; 3 Queen’s University, Kingston, ON, Canada; 4 University of North Carolina, Chapel Hill, NC; 5 Hematology, University of Pennsylvania, Philadelphia, PA.

The formation of antibodies (or inhibitors) to FVIII is a major complication in the treatment of humans with hemophilia A (HA), affecting up to 30% of individuals with severe or moderate disease Inhibitors develop mainly in young boys, and render the treatment with infused protein ineffective Although liver-directed gene therapy has been used to deliver therapeutic transgenes and can induce tolerance to the expressed protein, to date there have been no large animal studies using liver gene therapy to eradicate inhibitors to FVIII

We hypothesize that sustained expression of cFVIII will eradicate inhibitors to canine FVIII in dogs that have a history of inhibitors, thus demonstrating a potential alternative to the current immune tolerance