Luo FengTakeshi Hirota

Tsuyoshi Takara

RNtein Biotech Co., LtDTokyo, Japan

RNTein Biotech. Lab. Dedicated to human cancer gene therapy

San Gabriel, California, USA

AJS: a lethal weapon to combat with cancers Treatment of Human Cancers Using Mechanism-Based Lentivirus

Self-sufficiency in growth signals

Limitless replicative potential

Sustained angiogenesis

Evading apoptosis

Insensitivity to anti-growth signals

Tissue invasion & metastasis

The development of human cancer cells

Cdc6 is essential in the initiation of DNA replication:

A). Assembling pre-replication complex (pre-RC) with ORC, and loading MCMs to replication origins;

B). Licensing DNA replication to ensure one round DNA replication per cell cycle.

The novel function of AJSFL1 is the silencing of human Cdc6, the specific genes for cell proliferation: Specific Cdc6 shRNAs produced from the viral vector Inhibit Cdc6 and blockCancer cell proliferation, but not normal cells.

Cdc6 knockdown

Cdc6 shRNA

Cdc6

TH

TD

6Ai

TH

TP

CHLA255LA-N-2

TH

TP

TH

TD

6Ai

Mo

ck

Mo

ck

TH

TP

TH

TD

6Ai

MCF7

-actin

1 2 3 4 5 6 7 8

G1

S

G2

M

STOP

Recombinant LentivirusAJS2001

Adapted from J. M. Sedivy, Cancer Cell, 11:389-391, 2007

Oncogene activation

Telomere dysfunction

DNA damage response

p53

Apoptosis

Senescence

Rb

p16INK4a

p21

? Rb-E2F

Rb-E2FP

Cdk4/Cdk6

Cyclin D1

RbP E2F

SG1

Cdc6

1 2 3 4

Phosph-Rb-C Ser780

Phosph-Rb-C Ser807/811

IgG, light chain

— IP/β-p16INK4a

— — THTD — — — IP/mouse IgG

IgG, light chain

Phosph Rb-C Ser 807/811

1 2 3 4

1:5

1:10

1:25

IP/β-p16INK4a

— — — THTD

Reactivation of p16INK4a CDK inhibitory activity by Cdc6 knockdown

MCF7

MCF7/THTP

MCF7/THTD6Ai

Senescence-associated β-glactosidase (SA-β-Gla) positive staining caused by Cdc6 knockdown

Anti-H3K9me3/me2indirect fluorescentstaining

THTD6AiMock

Mock THTD6Ai

Confocal fluorescent microscopy

The formation of senescence associated heterochromatic foci viaCdc6 knockdown

　

A male patient age 55, with advanced gastric carcinoma found in April 2006 is first survivor by GFL1(AJS). After second injection since 07/06/2006, he gained weight with good appetite, and left hospital on 27/06/2006. After 4th injection, his gastric carcinoma disappeared without side effect. Now, 2 years and 8 month has passed since recovery, and he leads normal life with good appetite and feeling.

Speech about his own recovery experience at Cancer Control Association. He is now doing volunteer activity for many other cancer patient.

He and his wife filled with joy • • • has become active for social work

An advanced gastric carcinoma patient shows his recovery after receiving AJS injections.

Firs t in jec t ion on 24/04/2006

2 weeks a f t er 4 t imes of weekly in jec t ion on 05/06/2006

Multiple gastric scirrhous carcinoma of the male patient age 55

Emergency medica l admis s ion on 10/04/2006

　

Effect of AJS treatment shown by endoscope

A female patient age 52, who had major operation for uterus carcinoma metastated to liver and brain resulted in 14 days life expectancy because brain carcinoma remain, has amazingly recovered by continuous AJS injection for 3 month. Now, she leads pleasant normal life

Healthy expression after recoveryMRI of liver before injection

The treatment of a patient with brain metastatic ovary tumour

CT of brain after recoveryCT of brain before injection

A male patient age 64, recovered from parotid carcinoma metastated to lung and larynx. He had the therapy by day return to keep his office work. He has been nominated to chairman of I (AJS) patient association. He has now 6 grandchild, and is doing volunteer activity to promote AJS.

Before injection After injection

A female patient age 52, has recovered from breast cancer metastated to lymph node by only 4 times injection without surgery. In addition, her hair returned deep black. She is now doing volunteer activity for many other breast cancer patient by saying “do not need surgery, do not give up hope, breast cancer can be cured by injection only”.

A breast carcinoma patient voluntarily wants to have AJS treatment, not surgery.

Current recovery

After 1st injection After 2nd injection After 3rd injection

After 4th injection

Before injection

Her black hair