439 oncolytic newcastle disease virus iraqi virulent strain induce apoptosis in vitro through intrinsic pathway and association of both intrinsic and extrinsic pathways in vivo

439 Oncolytic Newcastle Disease Virus Iraqi Virulent Strain Induce Apoptosis In Vitro through intrinsic Pathway and Association of Both Intrinsic and Extrinsic Pathways In Vivo Molecular Therapy Volum[.]

Molecular Therapy Volume 23, Supplement 1, May 2015

CanCer – OnCOlytiC Viruses i

Systemic IFN has demonstrated some success in the treatment

of EAC, but its use is limited by systemic side effects Our vectors

provide a way of delivering IFN locally to the tumor Here, the

Cox2-IFN virus demonstrated a strong cytocidal effect at early time points in

all tested cell lines In fact, it caused significantly more cell death than

an otherwise identical vector expressing luciferase (p<0.05), thereby

demonstrating the profound effect of IFN expression Additionally,

there was a dose-dependent increase in cytocidal effect when the

Cox2-IFN virus was combined with cisplatin and radiation In vivo,

that same tumor-specific IFN-expressing vector demonstrated a

profound anti-tumor effect when compared to the groups treated with

a wild type virus and a saline control Replication dependent reporter

expression (luciferase) was measured to assess the viral replication

in the patient-derived tissue slices Both the Cox2-controlled vector

and the vector lacking a promoter showed strong replication in the

EAC tissue slices as expected Importantly, in the normal esophagus

samples, there was significantly less replication in the Cox2-controlled

group (p<0.05) Viral copy number (using the E4 primer) was also

used to quantify viral replication Here, a similar trend was observed

as there was virtually no replication of the Cox2 virus within the

normal tissue samples

We have demonstrated that a CRAd driven by the Cox2 promoter

has a strong oncolytic effect both in vivo and in vitro Moreover, our

Cox2-controlled vector has demonstrated cancer-specific replication

We believe that our novel vector possesses both therapeutic efficacy

and specificity, which makes it an ideal candidate for clinical

translation

437 Biosafety After the Injection of Carrier Cells

Infected With Oncolytic Adenovirus

Katsuyuki Hamada,1 Kazuko Takagi,1 Hiroshi Itoh,2 Kenzaburo

Tani,3 Akihiro Nawa.1

1 Obstetrics and Gynecology, Ehime University, Toon, Ehime,

Japan; 2 Animal Medical Center, Tokyo University of Agriculture

and Technology, Fuchu, Tokyo, Japan; 3 Advanced Molecular and

Cell Therapy, Kyusyu University, Higashi-ku, Fukuoka, Japan.

Although replication-competent viruses have been developed to

treat cancers, their cytotoxic effects are insufficient, since infection

with them is inhibited by generation of neutralizing antibodies To

address this limitation, we developed a carrier cell system to deliver

a replication-competent adenovirus Carrier cells infected with

oncolytic adenovirus were injected into syngeneic subcutaneous

ovarian tumors after immunization with adenovirus and induced

complete tumor regression by the induction of antiadenoviral and

antitumoral CTL responses To start clinical trial, toxicity and

biodistribution study were carried out in nude mice, rabbits and

beagle dogs Acute toxicity and distribution test were carried out

after the single injection of carrier cells into ovarian tumor in nude

mice Chronic toxicity test was carried out by 8 injections of carrier

cells into rabbits for 4 weeks Excretion study was carried out to

determine whether oncolytic adenovirus was excreted from the beagle

dogs after the single injection of carrier cells Acute toxicity test

did not show any lethal side effects in nude mice In biodistribution

test, single injection of carrier cells into ovarian tumor induced the

peak levels of oncolytic adenovirus the next day but did not show

any significant levels of that in nude mice 14 days after injection In

chronic toxicity test, 8 injections of 1.25×107 cells/kg or less did not

show any significant toxicity in rabbits In excretion test, oncolytic

adenovirus was not excreted into the urine and the stool of beagle

dogs This oncolytic adenovirus-infected carrier cell system might

prove effective and safe in the preclinical efficacy and the biosafety

test, respectively Clinical trial is being scheduled to treat recurrent

solid cancers

438 Oncolytic Immunotherapy for Treatment

of Breast Cancer, Including Triple-Negative Breast Cancer

Simona Bramante,1 Anniina Koski,1 Ilkka Liikanen,1 Minna Oksanen,1 Timo Joensuu,2 Sari Pesonen,1 Akseli Hemminki.1

1 Cancer Gene Therapy Group, Department of Pathology and Transplantation Laboratory and Haartman Institute, University of Helsinki, Helsinki, Finland; 2 Docrates Cancer Center, Helsinki, Finland.

Breast cancer is a heterogeneous disease, characterized by several distinct biological subtypes associated with specifical biological behavior and different clinical outcomes Triple-negative breast cancer (TNBC) is defined as estrogen receptor (ER)-negative, progesterone receptor (PR)-negative and lacking overexpression

of human epidermal growth factor receptor 2 (HER2) Among all the breast cancer subtypes, TNBC (10-15% of breast cancers) is associated with a worse prognosis

Oncolytic virus replication is an immunogenic phenomenon, and viruses can be armed with immunostimulatory molecules Cyclophosphamide (CP) is a chemotherapy drug that works by slowing or stopping cell growth At higher doses, CP is associated with increased cytotoxicity and immunosuppression, while at low continuous dosage it shows immunostimulatory and antiangiogenic properties Therefore, the combination of oncolytic immuno-virotherapy with low-dose CP is an appealing approach

We investigated the potency of oncolytic adenovirus Ad5/3-D24-GMCSF on a TNBC cell line and animal models, in combination with low-dose CP or its main active metabolite 4-hydroxycyclophosphamide (4-HP-CP) Furthermore, we summarized the breast cancer-specific human data from the Advanced Therapy Access Program (ATAP) Low-dose CP effectively increased the efficacy of Ad5/3-D24-GMCSF in vitro and in a TNBC xenograft mouse model In ATAP, treatments appeared safe and well-tolerated Fourteen out of sixteen breast cancer patients treated were evaluable for possible therapy benefit with modified RECIST criteria: one patient had minor response, four had stable disease, and nine had progressive disease One patient was alive at 941 days after treatment

Ad5/3-D24-GMCSF in combination with low-dose CP showed promising efficacy in preclinical studies and possible antitumor activity in breast cancer patients refractory to other forms of therapy This preliminary data supports continuing the clinical development

of oncolytic adenoviruses for treatment of breast cancer, including TNBC

439 Oncolytic Newcastle Disease Virus Iraqi Virulent Strain Induce Apoptosis In Vitro through intrinsic Pathway and Association of Both Intrinsic and Extrinsic Pathways In Vivo

Ahmed M Al-Shammari,1 Tagreed J Humadi,2 Eman H Al-Taee,2 Shalal M Al-Atabi,1 Nahi Y Yaseen.1

1 Experimental Therapy, Iraqi Center for Cancer and Medical Genetic Reseach, University of Mustansiriyah, Baghdad, Iraq;

2 Pathology, College of Veterinary Medicine, Baghdad University, Baghdad, Iraq.

Newcastle disease virus is very promising antitumor agent Iraqi virulent strain showed pronounced oncolytic activity This study aimed to investigate the specific pathway of apoptosis induced by Iraqi strain of Newcastle disease virus (NDV) on tumor cells In vitro and

In vivo Several cell lines were tested (Human Rhabdomyosarcoma cell line (RD), Human Glioblastoma multiforme culture (AMGM) and Murine mammary adenocarcinoma cell line ( AMN3) for ability

of Iraqi NDV strain to induce apoptosis and to give deep look about the main pathway that induce apoptosis in tumor cell lines by the

Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy

S174

Hematologic and immunologic diseases ii

virus The cell lines were infected with Newcastle disease virus and

investigated for apoptotic pathway by using immunocytochemistry for

caspase 3, 8, and 9 The result of this in vitro experiment revealed that

NDV induce apoptosis in tumor cell lines when compared to control

group with marked increase in the percentage of cells expressing

caspase 9 with low percentage of cells expressing caspase 8 in NDV

treated group at 24,48hr These results indicated the role of NDV

Iraqi strain in inducing apoptosis through intrinsic mitochondrial

pathway in vitro The in vivo experiment included female mice

transplanted with mammary adenocarcinoma transplantable tumor

line (AM3), after tumor growth; tumor bearing mice randomly

divided into groups, first group treated by NDV by intratumoral

injection, second group treated by NDV intrapretonially Caspase 3,

caspase 8, and caspase 9 expression in tumor sections were checked

after 24hrs, 48hrs, 72hrs, 7 and 14 days of treatment with NDV The

results revealed that NDV induce apoptosis significantly in treated

tumors by both ways IT and IP when compared with control group

There is significant (p<0.05) marked increase in the mean percentage

of cells expressing caspase 9 in NDV treated groups compared with

mean percentage of cells expressing caspase 8 and untreated control

group at 24,48,72,1week, 2 weeks post single injection treatment

Moreover, Caspase-8 expression in IT and IP groups was significantly

higher than control untreated group These results revealed that NDV

had a powerful effect on inducing mitochondrial (intrinsic) apoptotic

pathway in vivo in association of extrinsic pathway This association

can explained by NDV induction for antitumor immune response and

this immune response can trigger tumor killing by extrinsic pathway

The current work suggest that Iraqi oncolytic virulent strain of NDV

induce apoptosis through intrinsic pathway directly as the results of in

vitro study and induce extrinsic pathway indirectly through immune

stimulation properties of NDV

Hematologic and Immunologic Diseases II

440 Optimization of RNA Trans-Splicing for the

β-Globin Gene; Detecting Trans-Splicing Events

Targeting Endogenous β-Globin Pre-mRNA in

Human Erythroid Cells

Naoya Uchida,1 Charlotte Platner,1 Josiah Ballantine,1 Matthew M

Hsieh,1 Brian Mozer,2 Lloyd G Mitchell,2 Kareem N Washington,3

John F Tisdale.1

1 MCHB, NHLBI/NIDDK, NIH, Bethesda, MD; 2 RetroTherapy,

Bethesda, MD; 3 Genetics and Human Genetics, Howard

University, Washington, DC.

Sickle cell disease is caused by a point mutation in exon 1 of the

β-globin gene (3 exons and 2 introns) RNA splicing between two

distinct pieces of pre-mRNA, known as trans-splicing, represents a

potential therapeutic strategy allowing replacement of the mutated

exon 1 with a normal exon Induction of exogenous mRNA splicing

using spliceosome-mediated splicing requires an RNA

trans-splicing molecule (RTM) which imitates endogenous cis-trans-splicing

elements However, clinical application of trans-splicing for globin

disorders will require high efficiency, thus we sought to optimize the

efficiency of trans-splicing targeting the β-globin gene to replace the

exon 1 among human erythroid cells

To optimize the binding domain targeting the β-globin gene, the

randomized binding domains of 20-600b were generated by sonicating

the β-globin gene, and these were inserted into RTM-expressing

plasmids downstream of the 5’ half of GFP and a 5’ splice site In

addition, we designed a target plasmid which encodes the β-globin

gene and an artificial 3’ splice site connected to 3’ site of the other half

of GFP In transfection of both RTM and target plasmids, we selected 6

candidates from several thousand RTMs, which produced the brightest GFP-positive cells (maximal trans-splicing), and interestingly, all 6 binding domains targeted the β-globin intron 2

To evaluate efficiency of trans-splicing for human endogenous β-globin RNA, we constructed lentiviral vectors encoding RTMs which contain the γ-globin cDNA connected to a 5’ splice site and 3 candidates (1E1; 0.6kb, 2D10; 0.7kb, and 13-1; 0.8kb) of β-globin binding domains (selected from the 6 candidates) Human CD34+ cells were transduced with these RTM-encoding lentiviral vectors,

and these cells were differentiated to erythroid cells in vitro

Trans-splicing was detected by RT-PCR and sequencing in RTM 2D10 and 13-1 but not in RTM 1E1, suggesting that longer binding domains improve trans-splicing efficiency To elongate the binding domain

in RTM 13-1, we designed RTM BGin2 (containing 5’ splice site of β-globin intron 2; 0.9kb) and BGex1-in2 (containing whole β-globin sequence except exon 3; 1.3kb) In both RTM BGin2 and BGex1-in2, ∼4-fold higher trans-splicing was detected by RT-qPCR than RTM 13-1, which resulted in ~0.07% trans-splicing efficiency as compared to endogenous human β-globin RNA, implying that the 5’ splice site of intron 2 should be included as a binding domain for efficient trans-splicing

In summary, we developed a screening system to select more efficient binding domains for trans-splicing For the first time, we obtained detectable levels of spliceosome-mediated trans-splicing (~0.07%) for endogenous β-globin RNA in human erythroid cells which were transduced with RTM-expressing lentiviral vectors Further optimization is required to develop trans-splicing based gene therapy

441 Direct Comparison of EF1α and PGK Promoters Reveals Superior Performance of the PGK Promoter for Expression of the Common Gamma Chain in a Canine Model of In Vivo Foamy Virus Gene Therapy for Severe Combined Immunodeficiency

Christopher R Burtner,1 Humbert Olivier,1 Patricia O’Donnell,2 Nicholas Hubbard,3 Daniel Humphrys,1 Jennifer E Adair,1,4 Grant

D Trobridge,5 Troy R Torgerson,3,4 Andrew M Scharenberg,3,4 David J Rawlings,3,4 Peter J Felsburg,2 Hans-Peter Kiem.1,4

1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 2 School of Veterinary Medicine, University

of Pennsylvania, Philadelphia, PA; 3 Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA; 4 Department of Medicine, University of Washington, Seattle, WA; 5 Department of Pharmaceutical Sciences, Washington State University, Pullman, WA.

Foamy viruses (FV) are ideal vectors for in vivo gene therapy because FV infection is non-pathogenic in people, FV are more resistant to serum inactivation than lentiviruses packaged with the vesicular stomatitis virus glycoprotein (VSV-G), and FV exhibit favorable proviral integration patterns compared to gammaretroviruses In vivo gene therapy with a FV vector expressing the human common gamma chain (γC) under the short EF1α promoter results in a functional immune reconstitution in X-SCID dogs However, T cell reconstitution occurred through the correction

of a limited number of progenitors, possibly due to sub-therapeutic expression of the transgene from the EF1α promoter To address this,

we compared the efficacy of the EF1α and PGK promoters in a direct head-to-head competitive experiment by injecting two X-SCID pups with equal titers of the EF1α and PGK vectors marked with different fluorophores

A significant population of gene-marked lymphocytes appeared in the PGK arm 42 days post in vivo gene therapy, which continued to expand over time (Fig 1A) By 84 days post injection, lymphocyte