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




                          Luo Feng
                        Takeshi Hirota
                       Tsuyoshi Takara


                     RNtein Biotech Co., LtD
                          Tokyo, Japan


                      RNTein Biotech. Lab.
             Dedicated to human cancer gene therapy
                   San Gabriel, California, USA
Self-sufficiency      Sustained
       in growth signals     angiogenesis        Insensitivity to
                                               anti-growth signals




Limitless replicative   Evading apoptosis   Tissue invasion
      potential                              & 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 block
 Cancer cell proliferation, but not normal cells.




                                  LA-N-2                         MCF7              CHLA255
      Cdc6 shRNA




                                                                                          THTD6Ai
                                                THTD6Ai




                                                                         THTD6Ai


                                                                                   THTP
                                                          Mock
                                  Mock

                                         THTP




                                                                  THTP
                                                                                                    Cdc6
Cdc6 knockdown
                                                                                                    -actin

                                   1     2       3          4       5        6        7     8
M
Recombinant                       G1
 Lentivirus
  AJS2001     G2
                           STOP




                   S
Cdc6
          Telomere                       Oncogene
         dysfunction                     activation
                                                              ?                        Rb-E2F
                                                                          Cyclin D1
                                                                          Cdk4/Cdk6


                        DNA damage                         p16INK4a              P    Rb-E2F
                         response




                 p53          p21                                     P   Rb           E2F
                                         Rb



        Apoptosis


                                    Senescence                                 G1              S



Adapted from J. M. Sedivy, Cancer Cell, 11:389-391, 2007
Reactivation of p16INK4a CDK inhibitory activity by Cdc6 knockdown


            — —                       
              
            THTD — —                   —       IP/mouse
            —IgG                              IP/β-
            p16 INK4a
                                                Phosph-Rb-C Ser780


                                                Phosph-Rb-C Ser807/811


                                                IgG, light chain

              1      2      3      4



               — —              —          
               THTD
                     1:10

                            1:25
               1:5




                                                IP/β-
                                       p16 INK4a
                                                   Phosph Rb-C Ser 807/811
                                                 IgG, light chain
               1     2      3          4
Senescence-associated β-glactosidase (SA-β-Gla)
  positive staining caused by Cdc6 knockdown

   MCF7




                MCF7/THTP




                               MCF7/THTD6Ai
The formation of senescence associated heterochromatic foci via
                       Cdc6 knockdown



                                    Anti-H3K9me3/me2
                                    indirect fluorescent
                                    staining


        Mock          THTD6Ai




                                    Confocal fluorescent
                                    microscopy




        Mock          THTD6Ai
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.




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

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


                 An advanced gastric carcinoma patient shows
                  his recovery after receiving AJS injections.
Multiple gastric scirrhous carcinoma of the male patient age 55

            Emergency medical admission on 10/04/2006


 




                                                   First injection
                                                   on
                                                   24/04/2006




                                     2 weeks after 4 times of weekly
                                     injection on 05/06/2006




                           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




          CT of brain before injection                         CT of brain after recovery




                      MRI of liver before injection         Healthy expression after recovery

            The treatment of a patient with brain metastatic ovary tumour
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”.


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




                            After 4th injection           Current recovery            Her black hair


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

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

  • 1. AJS: a lethal weapon to combat with cancers Treatment of Human Cancers Using Mechanism-Based Lentivirus Luo Feng Takeshi Hirota Tsuyoshi Takara RNtein Biotech Co., LtD Tokyo, Japan RNTein Biotech. Lab. Dedicated to human cancer gene therapy San Gabriel, California, USA
  • 2. Self-sufficiency Sustained in growth signals angiogenesis Insensitivity to anti-growth signals Limitless replicative Evading apoptosis Tissue invasion potential & metastasis The development of human cancer cells
  • 3. 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.
  • 4. 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 block Cancer cell proliferation, but not normal cells. LA-N-2 MCF7 CHLA255 Cdc6 shRNA THTD6Ai THTD6Ai THTD6Ai THTP Mock Mock THTP THTP Cdc6 Cdc6 knockdown -actin 1 2 3 4 5 6 7 8
  • 5. M Recombinant G1 Lentivirus AJS2001 G2 STOP S
  • 6. Cdc6 Telomere Oncogene dysfunction activation ? Rb-E2F Cyclin D1 Cdk4/Cdk6 DNA damage p16INK4a P Rb-E2F response p53 p21 P Rb E2F Rb Apoptosis Senescence G1 S Adapted from J. M. Sedivy, Cancer Cell, 11:389-391, 2007
  • 7. Reactivation of p16INK4a CDK inhibitory activity by Cdc6 knockdown — —    THTD — — — IP/mouse —IgG    IP/β- p16 INK4a Phosph-Rb-C Ser780 Phosph-Rb-C Ser807/811 IgG, light chain 1 2 3 4 — — —  THTD 1:10 1:25 1:5  IP/β- p16 INK4a Phosph Rb-C Ser 807/811 IgG, light chain 1 2 3 4
  • 8. Senescence-associated β-glactosidase (SA-β-Gla) positive staining caused by Cdc6 knockdown MCF7 MCF7/THTP MCF7/THTD6Ai
  • 9. The formation of senescence associated heterochromatic foci via Cdc6 knockdown Anti-H3K9me3/me2 indirect fluorescent staining Mock THTD6Ai Confocal fluorescent microscopy Mock THTD6Ai
  • 10. 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. He and his wife filled with joy • • • has become active for social work Speech about his own recovery experience at Cancer Control Association. He is now doing volunteer activity for many other cancer patient. An advanced gastric carcinoma patient shows his recovery after receiving AJS injections.
  • 11. Multiple gastric scirrhous carcinoma of the male patient age 55 Emergency medical admission on 10/04/2006   First injection on 24/04/2006 2 weeks after 4 times of weekly injection on 05/06/2006 Effect of AJS treatment shown by endoscope
  • 12. 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 CT of brain before injection CT of brain after recovery MRI of liver before injection Healthy expression after recovery The treatment of a patient with brain metastatic ovary tumour
  • 13. 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
  • 14. 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”. Before injection After 1st injection After 2nd injection After 3rd injection After 4th injection Current recovery Her black hair A breast carcinoma patient voluntarily wants to have AJS treatment, not surgery.