Trial Information

Phase I/II Study of EBV-LMP1 Targeted DNAzyme in Nasopharyngeal Carcinoma

INTRODUCTION Nasopharyngeal carcinoma (NPC) is a serious health problems worldwide,
particularly in the southern Chinese population, with an incidence rate ranging from 15 to
50 per 100 000. NPC is an epithelial malignancy with a striking racial and geographic
distribution differences. High incidence rates are observed in the southeast Chinese
population, and similar rates have been reported in these people wherever they have
migrated, including Singapore, Taiwan, and Hong Kong. This incidence is almost 100 fold
higher than in white populations. The most unique feature of NPC is its almost universal
association with the infection of Epstein-Barr virus (EBV), which is the first human virus
identified to be involved in the pathogenesis of several malignancies and has a particularly
close association with NPC, as EBV genome can be detected in virtually all NPC cells. While
radiotherapy has been the first-line treatments for NPC, radio-resistance remains a
significant clinical issue for the NPC radiotherapy. Thus, there is unmet medical needs to
discover and develop novel radiosensitizers for NPC therapy.

EBV infection in NPC is classified as type II latent infection in which only EBV nuclear
antigen-1(EBNA-1), latent membrane protein-1(LMP1), LMP2, and EBV early RNA (EBER)
expressions can be detected. Among these proteins, LMP1 is thought to play a key role in the
pathogenesis of NPC. As a 60kD integral membrane protein, LMP1 functions as a constitutively
active tumor necrosis factor receptor (TNFR), and contributes to multiple aspects of NPC
through activating a number of signaling pathways including nuclear factor NF-κB, activator
protein-1(AP-1), and Janus kinase/signal transducer and activator of
transcription(JAK/STAT). Activation of NF-kB or AP-1 by LMP1 has been linked to the
upregulation of some cellular proteins and inhibition of apoptosis. Depending on the cell
types, expression of LMP1 has been shown to play different roles in response to biological
and physiological stimulus. It acts as a primary oncoprotein for human cell immortalization
and is also shown as the only EBV-coded product that can transform rodent fibroblast cell
line, human epithelial cells and keratinocytes.

Given the critical role of viral oncoproteins in transformation and apoptosis, suppression
of some viral oncoproteins would provide a sensible strategy to genetically treat NPC.
Indeed, antisense oligonucleotides against LMP1 or EBNA1 have been shown to inhibit viral
oncoprotein expression, induce apoptosis, and sensitize the EBV-positive cells to cytotoxic
agents. Recently, some experimental studies indicated that the RNA interference against LMP1
exhibited an anti-proliferative and anti-metastasis effect in LMP1 expressing NPCs. These
results suggested that EBV-encoded LMP1 may present a potential molecular target for
treatment of EBV-associated carcinomas.

DNAzymes are synthetic, single-stranded DNA oligonucleotides that can be engineered to bind
to their complementary sequence in a target messenger RNA (mRNA) through Watson-Crick base
pairing and cleave the mRNA at predetermined phosphodiester linkages. A general model for
the DNAzyme has been proposed, and is known as the ''10-23'' model. A ''10-23'' DNAzyme has
a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains
of seven to nine deoxyribonucleotides at each arm. In vitro analyses showed that this type
of DNAzyme could effectively cleave its substrate RNA at purine: pyrimidine junctions under
physiological conditions. These agents have been used in a number of in vitro and in vivo
applications to inhibit the expression of their target genes and the dependent genes. Their
capacity to block development of a diverse range of pathologies in animal models suggests
that DNAzymes can be used as therapeutic agents.

To develop EBV-LMP1 targeted DNAzymes for NPC treatment, we showed that the
phosphorothioate-modified ''10-23'' DNAzymes specifically targeted at the LMP1 mRNA could
significantly down-regulate the expression of LMP1 in a nasopharyngeal carcinoma cell (NPC)
and affected the down-stream pathways activated by LMP1, including the NF-κB pathway. It was
also demonstrated that suppression of the LMP1 expression by the LMP1-targeted DNAzyme DZ1
could enhance radiosensitivity both in vivo and vitro. Radio-resistance has been one of the
impediments in clinical settings for effective cancer therapy, which is thought to be
associated with multiple signaling pathways in different cancer types. ATM (ataxia
telangiectasia mutated) is a nuclear 350-kDa protein kinase with a carboxylterminal
phosphatidylinositol 3-kinase-like kinase domain[1]. It functions as a member of a
coordinated system that detects DNA breaks; arrests the cells temporarily at G1, S, or G2
checkpoints; and activates DNA repair. Cells lacking functional ATM protein show increased
sensitivity to ionizing radiation (IR) and other genotoxic events. NF-κB (nuclear factor
kappa B) can activate a great number of genes involved in stress responses, inflammation,
and programmed cell death (apoptosis). P50 homodimers or p50/p65 or p50/c-Rel heterodimers
bind to the NF-κB DNA binding sites in the promoter regions of many stress-responsive genes,
suggesting a complex gene and physiological regulation network controlled by NF-κB in stress
response. The elevated basal NF-κB activity in certain cancers has been linked to tumor
resistance to chemotherapy and radiation. Inhibition of NF-κB blocked the adaptive
radioresistance. Our studies for the molecular mechanism of the LMP1-DNAzyme mediated
radiosensitization revealed that LMP1 activated the ATM expression through the NF-κB pathway
and inhibition of LMP1 expression by the DNAzyme attenuated the binding of NF-κB
transcription factor to the ATM promoter. Further evidence showed that the radiosensitivity
was recovered when the ATM expression was knocked down by siRNA in NPCs. Together, all our
data support our hypothesis and provide solid experimental basis for the use of
LMP1-targeted DNAzymes as potential radiosensitizers for treatment of the EBV-associated
carcinomas.

Toxicological studies in mice showed that no morbidity or mortality was observed in any of
the dosing groups during the course of the study (50mg, 100mg, and 200mg/kg). All
hematological values and biochemistry results from tests of hepatic and renal function were
normal. No microscopic lesion that could be attributed to the modified DNAzyme
oligonucleotide treatment was found in liver, spleen and kidney in any groups. After i.v.
administration of 100 mg/kg DNAzyme oligonucleotide in mice, the peak plasma concentration
of 24.13±2.6μg/ml was achieved. The decrease in plasma concentration of DNAzyme followed a
bi-exponential pattern with initial distribution half-life (t1/2α) of 0.18±0.03 h and a
terminal half-life (t1/2β) of 2.55±1.0 h, and area under the plasma concentration-time curve
(AUC) was 54.17±9.1μg.h/ml.

STUDY DESIGN This study will be a randomized, double-blinded and placebo controlled Phase
I/II clinical trial. Forty (40) patients will be randomized to one of two groups of equal
size: placebo group receiving saline by intra-tumor injection and standard radiotherapy; or
DZ1 group receiving LMP1 DNAzyme (DZ1) and standard radiotherapy. The placebo group will
provide the basis for assessment of safety and efficacy of DZ1.

Patients receive placebo or DZ1 injection two (2) hours prior to radical radiation therapy
on Monday and Thursday over seven weeks. The radical radiotherapy is given to patients 5
times per week with 2 Gy of each treatment. The entire procedure lasts seven weeks.

All patients will complete the study at 104 weeks post-first injection. The patients will
undergo assessment and testing every week in the first seven weeks, then every three months
from the weeks 8 to week 104.

The study will include evaluations of safety and tolerability: