565 A Placebo Controlled Phase IIb Clinical Studyof TissueGene C (TG C) in Patients with Osteoarthritis Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & C[.]
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DNA VECTOROLOGY & GENE TARGETING II
provide a feasible strategy to design and make novel Ad vector for
vaccine development, which will avoid spending too much time and
cost to screen new Ad vectors based on the traditional way
563 Construction of a Novel Bicistronic
Adenoviral Vector
Feilong Jie,1 Shengyao Wang,2 Jie Luo,1 Na Zhang,3 Jingang
Zhang,3 Hongwei Li.1
1 Southern Medical University, Guangzhou, Guangdong,
China; 2 Mulan Pharmaceuticals, Shenzhen, China; 3 Institute of
Transfusion Medicine, The Academy of Military Medical Sciences,
Beijing, China.
Achieving consistent expression of two therapeutic proteins in
the same cells is an important issue for gene therapy applications
Currently, the most commonly employed strategies include the use of
multiple promoters, or internal ribosome entry site (IRES) elements
However, it has shown that the protein encoded downstream of the
IRES in these vectors is not reliably expressed Meanwhile, the
use of multiple promoters may be compromised by interference
between promoters, promoter silencing, and vector rearrangements
or deletions We have constructed a novel adenoviral vector,
pShuttle-CMV-GFP-2A-Cherry, with a shared CMV promoter and a 2A peptide
encoding region from the foot-and-mouth disease virus (FMDV),
which cleaves at its own C-terminus, thereby separating the “fusion
protein” into two independent proteins Importantly, the restriction
enzyme Apa I site at the 5’ end is designed to help replace Cherry
with target proteins, resulting in the C site of cleaved fusion protein
with an excess proline In our study, 293T cells were separately
transfected by the calcium phosphate method with the plasmids
CMV-GFP-2A-Cherry, CMV-GFP, or
pShuttle-CMV-Cherry while 293A cells were separately infected with Ad5-
CMV-GFP-2A-Cherry, Ad5-CMV-GFP or Ad5-CMV-Cherry We
found that GFP or Cherry expression delivered by related plasmids
or packaged viruses were almost at the same level no matter the cell
type It indicates that the novel adenoviral vector can elicit bicistronic
expression simultaneously and equivalently Moreover, the C end of
cleaved fusion protein has only one excess amino acid of proline
These results will greatly benefi t the study on both structure and
function of target protein and gene therapy
564 Ciprofl oxacin Regulates Negatively
Egr-1 Transcriptional Activity in Human Primary
Tenocytes Transduced with Ad-Egr-1/Luc
Francisco Martinez-F,1,2 Araceli Barrera-Lopez,1 Hugo
Sandoval-Zamora,1 Karina Guzman-M,1 Alejandro Jimenez-Orozco,2
Christian Y Lomeli-R,1 David T Curiel,3 Rebecca E
Franco-Bourland.4
1 Molecular Biotherapeutic Program, Skin & Tissue Bank,
National Institute of Rehabilitation Ministry of Health, Mexico
City, DF, Mexico; 2 Dapartment of Pharmacology, School of
Medicine, National Univeristy of Mexico, Mexico City, DF,
Mexico; 3 Department of Radiation Oncology, School of Medicine,
University of Washington., St Louis, MO; 4 Department of
Biochemistry, National Institute of Rehabilitation, Mexico.
Introduction: Tenocytes proliferation rate is an essential factor for
remodeling and maintenance of tendon integrity Negative balance of
the rhythm of cell proliferation is a key factor for infl ammatory process
and tendon rupture Helicases inhibitors such as fl uoroquinolones have
been reported as factor for tendon rupture Hereby, we analyze the
effect of ciprofl oxacin on rate of cell proliferation in human primary
tenocytes and the effect on the transcriptional regulation of egr-1
promoter based on transduction of adenoviral vector Adegr1-Luc
Materials and methods: Cells and adenoviral vectors: No replicative
recombinant adenovirus (AdEgr1-Luc) were packaged at large scale
in HEK-293 cells and purifi ed according to the current protocol based
on cesium chloride gradient protocol for in vivo application Viral Stock was titled by plaque assay and OD Human primary tenocytes (HPT) were obtained based on collagenase digestion protocol and cultured in DMEM/F12 Media supplemented with 10% HI-FBS and antibiotics under standard culture conditions for 1 week and stored for experimental procedure 5x104 Tenocytes were seeded/well After
12 hrs, cells were infected with AdEgr1-Luc at 50 MOI´s during two hrs in serum reduced media After infection, cells were keeped
in 1% of FBS for 24 hours at environment standard conditions for culture and ciprofl oxacin at 5 a 10 μg/ml (15, 30, 60, 90 and 120 seg) Protein extraction was performed at 2, 6 and 12 hrs based
on Cell Glo Lysis Buffer (Promega corp.) and luciferase activity was quantifi ed using a multidetector DTX-880 Results: Human tenocytes transduced with AdEgr1-Luc are positively responsive
to (10%) of Egr-1 promoter (18,233 LC/s) Luminescent activity
in presence of ciprofl oxacin was observed at 2, 6 and 12 hrs (1,490 LC/S; 1,704.66 LC/s and 1,851.33 LC/s, respectively) Conclussion Ciprofl oxacin inhibits transcriptional activity of egr-1 promoter in human primary tenocytes However, this fact is actually studies to determinate the signal transduction of regulation in tenocytes and the effect on cell proliferation rate by Cell proliferation assays Acknowledgments: This research project is granted by the National Council of Science and Technology of México Grant FOSIS/ CONACYT-Salud-2011-1-161624
DNA Vectorology & Gene Targeting II
565 A Placebo Controlled Phase IIb Clinical Studyof TissueGene-C (TG-C) in Patients with Osteoarthritis
Jung Jong Cho,1 Tae Won Kim,1 Yeo Myeong Park,1 Eu Gene Jeong,1 Moon Jong Noh,1 Kwan Hee Lee,1 Bum Sup Lee.1
1 Kolon Life Science, Inc., Gwacheon-Si, Gyeonggi-Do, Korea.
The objective of this study was to determine both safety and effi cacy of TG-C in patients with knee osteoarthritis (OA) TG-C is
a cell mediated gene therapy that contains non-transduced (hChonJ) and transduced (hChonJb#7) human allogeneic chondrocytes The hChonJb#7 cells were transduced with retrovirus encoding TGF-1 gene The study was a multicenter, randomized, single-blind, placebo-controlled phase IIb trial (NCT01671072) The OA patients (n = 54) were randomized into two groups; TG-C (n=27, 1.8x107cells, 3.5 ml/ knee) and placebo (n=27, normal saline, 3.5 ml/knee) TG-C or saline was injected directly to the knee joints and clinical evaluations were made at 12 and 24 weeks post treatment The primary evaluation endpoint was the changes of the International Knee Documentation Committee (IKDC) which measures pain, sports activities, and daily function Secondary evaluation endpoints were Western-Ontario and MacMaster University (WOMAC) score, Knee Injury and Osteoarthritis Outcome Score (KOOS), and 100 mm Visual Analogue Scale (VAS) Blood samples were analyzed to detect the replication competent retrovirus (at 12 and 24 weeks post treatment), TGF-1 DNA and protein (starting from 2 weeks up to 6 months post treatment) TG-C treatment showed signifi cant improvement in the primary and secondary clinical evaluations of IKDC (P <0.05) and
100 mm VAS (P <0.05) compared to the placebo as shown in table
1 Serum TGF-1 protein levels were within normal ranges (65ng/ ml) RCR and TGF-1 DNA were not detected at the completion of the study
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DNA VECTOROLOGY & GENE TARGETING II
Table 1 Changes in Scores of the Primary and the Secondary Evaluation Parameters
at 24 Weeks Post Treatment
(N=27) P values primary endpoint IKDC 16.39±15.63 8.05±11.18 P=0.0304
WOMAC -13.81±19.23 -7.50±13.02 P=0.1692
Secondary endpoint KOOS -22.96±27.15 -13.92±16.85 P=0.1508
VAS -24.83±27.29 -10.79±18.22 P=0.0321
In summary, the current Phase Iib study indicated that TG-C treatment
improved the primary and the secondary clinical evaluation criteria
for pain, sports activities, and quality of daily life in patients with
knee OA when compared to the placebo control
566 A Novel Oncolytic HSV Design Based on
the Common Over-Expression of miR-21 in Tumors
Marco Marzulli,1 Lucia Mazzacurati,1 Bonnie Reinhart,1 Mark E
Hatley,2 Joseph C Glorioso,1 Paola Grandi,3 Justus B Cohen.1
1 Microbiology and Molecular Genetics, University of Pittsburgh,
Pittsburgh, PA; 2 Oncology, St Jude Children’s Research Hospital,
Memphis, TN; 3 Neurological Surgery, University of Pittsburgh,
Pittsburgh, PA.
Oncolytic virus (OV) therapy is aimed at the selective destruction
of cancer cells without harming healthy tissue Typically, OVs contain
mutations that block lytic virus replication in normal cells but that are
complemented in cancer cells OVs derived from herpes simplex virus
1 (oHSV) have shown effi cacy in preclinical models of several types
of cancer and safety in Phase I human trials, but therapeutic outcomes
have been disappointing This is due in part to the attenuating
mutations in these viruses that provide tumor selectivity, but also
reduce the viral lytic replication activity in tumors Recent studies
have demonstrated that lytic virus replication can be brought under
the control of cellular microRNAs (miRNAs) that are differentially
expressed in normal and cancer cells MiRNAs negatively regulate
gene expression by binding to complementary mRNA targets
MiRNA expression profi les of cancer cells typically show both
up-regulated and down-up-regulated miRNAs, but each cancer type has a
unique miRNA signature showing dysregulation of different sets of
miRNAs Thus targeted therapies based on these unique signatures
are applicable only to the corresponding cancer type Remarkably,
studies of a wide range of cancer types have identifi ed one miRNA,
the oncomir miR-21, which is up-regulated nearly universally in
cancer cells For example, increased miR-21 expression has been
reported in 27 types of cancer These results and functional studies
are consistent with a key role for miR-21 in the development and/
or maintenance of the neoplastic state (e.g., Chan et al., Cancer Res
65:6029-33, 2005; Hatley et al., Cancer Cell 18:282-93, 2010; Ma
et al., PNAS 108:10144-9, 2011) We have sought to take advantage
of the common up-regulation of miR-21 in cancer cells by creating
a novel control circuit in which the host cell miRNA induces, rather
than represses, lytic HSV replication To accomplish this, we have
engineered a miR-21-responsive dominant negative (dn) version of
the essential HSV replication gene, UL9, into the HSV genome The
recombinant virus (KGdn9T21) showed replication comparable to
that of a control virus without dnUL9 gene in cancer cell lines such as
U2OS, A431, A549, and in mouse embryonic fi brobasts (MEFs) In
contrast, KGdn9T21 replicated very poorly compared to the control
in the absence of miR-21, such as in Vero cells and miR-21-/- MEFs
These results support the potential of this vector design to serve as
a safe and effective oHSV platform that provides a general level of
safety and effi cacy for the treatment of cancer
567 Comparing the Activity of TALENs Constructed with Different Guanine-Targeting RVDs
Yanni Lin,1 Thomas J Cradick,1 Eli J Fine,1 Gang Bao.1
1 Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA.
The ability of designing the modular DNA-binding domains of TAL Effector Nucleases (TALENs) has led to their effective use in genomic editing in a number of biological applications The DNA-binding domain of TALENs contains a tandem array of 33-35 amino-acid repeats Each repeat specifi es one nucleotide through the 12th and 13th amino acids, named the repeat-variable di-residues (RVDs) The RVDs NI, HD and NG specifi cally bind to nucleotides A, C, and T respectfully in the target sequence However, multiple RVDs, such as NN (Asn-Asn), NK (Asn-Lys) and NH (Asn-His) recognize nucleotides G with different levels of specifi city and binding affi nity The NN RVD binds to either G or A with similar effi ciency; the
NK RVD has higher specifi city but lower affi nity in binding to G compared with NN Although according to previous studies of TAL effectors, the NH RVD might have high affi nity and high specifi city
in binding to G, it is still unclear if TALENs constructed with NH will have higher on-target activity than those constructed with RVDs
NN or NK Here we report the comparison of TALENs constructed using NK, NH, and NN (KNH TALENs) respectively that target six specifi c DNA cleavage sites To investigate how the number of Gs
in the target sequence correlates with the relative activities of the use
of each RVD, we compared individual TALEN monomer activity
of KNH TALENs using a single-strand annealing assay We further quantifi ed the off-target effects of KNH TALENs The results from this study will provide insight into the selection of G-specifying RVDs based on both cleavage activity and target specifi city, and thus help optimize TALEN designs for genome engineering
568 In Vivo Cleavage of Transgene Donors Promotes Nuclease-Mediated Targeted Integration
Sandra Cristea,1 Yevgeniy Freyvert,1 Yolanda Santiago,1 Michael
C Holmes,1 Fyodor D Urnov,1 Philip D Gregory,1 Gregory J Cost.1
1 Sangamo BioSciences, Richmond, CA.
Targeted DNA integration is commonly used to eliminate position effects on transgene expression Integration can be targeted to specifi c sites in the genome via both based and homology-independent processes Both pathways start the integration process with a site-specifi c break in the chromosome, typically from a zinc-fi nger nuclease (ZFN) We previously described an effi cient homology-independent targeted integration technique that captures short (<100 bp) pieces of DNA at chromosomal breaks created by ZFNs We show here that inclusion of a nuclease target site on the donor plasmid followed by in vivo nuclease cleavage of both the donor and the chromosome results in effi cient integration of large, transgene-sized DNA molecules into the chromosomal double-strand break Successful targeted integration via in vivo donor linearization
is demonstrated at fi ve distinct loci in two mammalian cell types, highlighting the generality of the approach Finally, we show that CHO cells, a cell type recalcitrant to homology-based integration, are profi cient at capture of in vivo-linearized transgene donors Moreover, we demonstrate knockout of the hamster FUT8 gene via the simultaneous ZFN- or TALE nuclease-mediated integration of
an antibody cassette Our results enable effi cient targeted transgene addition to cells and organisms that fare poorly with traditional homology-driven approaches