Successful treatment of the murine model
    of cystinosis using bone marrow
           cell transplantation


           ...
Rational

   Cystinosis
       Genetic disease (CTNS gene)
       Gene expressed in every cells of the body
       Mul...
Adult bone marrow stem cells


               Adult bone marrow stem cells
                   Pluripotent
              ...
Experimental Design
Donors:
• GFP transgenic mice
• Ctns-/- mice                                           2 months in cul...
Confocal microscopy analysis of BMC and HSC
             Transplanted Mice
 Ctns-/- BMC-treated Ctns-/-   MSC-treated Ctns...
BMC-derived cells tissue integration
                                 IVIS imaging system




Ctns-/- mice transplanted
wi...
Cystine Content at 2 and 4 months post-transplant
                                           Spleen                     Li...
Cystine Content 4 months post-transplant
                                                      UCSD Biochemical Genetics l...
Long-term studies of BMC transplantation
           (Preliminary data)
              15 months-old mice treated for 7 mont...
Long-term studies of BMC transplantation
           (Preliminary data)
                                            15 mont...
Long-term studies of BMC transplantation
                  (Preliminary data)
                              15 months-old ...
Long-term studies of BMC transplantation
                                            (Preliminary data)
                  ...
Summary
4 months studies

         BMC and HSC
             Integration in all organs tested: kidney, liver, spleen, hea...
Relevance
     This work is a proof of concept for
autologous BMC or HSC transplantation for
         the treatment of cys...
Next steps
   Optimizing HSC transduction and test efficiency and
    safety of HSC transduced by scAAV1-CTNS on the
    ...
Acknowledgments



                                                           Frank Harrison
                             ...
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Successful treatment of the murine model of cystinosis using ...

  1. 1. Successful treatment of the murine model of cystinosis using bone marrow cell transplantation Stephanie Cherqui, Ph.D
  2. 2. Rational  Cystinosis  Genetic disease (CTNS gene)  Gene expressed in every cells of the body  Multisystemic disease  All the cells are progressively dying  Introduce the gene CTNS in all the cells of the body  Stem cells are the only vehicle capable of replacing most of the cells in the body
  3. 3. Adult bone marrow stem cells  Adult bone marrow stem cells  Pluripotent  Safe  Currently used in clinical trials  3 types :  Whole bone marrow cell (BMC)  Hematopoietic stem cells (HSC)  Mesenchymal stem cells (MSC)
  4. 4. Experimental Design Donors: • GFP transgenic mice • Ctns-/- mice 2 months in culture MSC progenitors BMC HSC MSC Irradiation Tail Vein Injection 2 month old Ctns-/- Recipients Analysis 2 months or 4 months after
  5. 5. Confocal microscopy analysis of BMC and HSC Transplanted Mice Ctns-/- BMC-treated Ctns-/- MSC-treated Ctns-/- WT BMC-treated Ctns-/- objective X 20 Nuclei (Dapi) GFP+ cells Muscle Brain Eye objective X 60 Actin (Phalloidin) Nuclei (Dapi) GFP+ cells
  6. 6. BMC-derived cells tissue integration IVIS imaging system Ctns-/- mice transplanted with luciferase expressing BMC Wildtype mice transplanted with luciferase expressing BMC
  7. 7. Cystine Content at 2 and 4 months post-transplant Spleen Liver Kidney 250 90 200 80 200 70 150 60 150 50 100 40 100 30 20 50 50 10 0 0 0 2 4 2 4 2 4 Heart Muscle months nmol half cystine/mg protein 90 45 80 40 70 35 60 30 50 25 40 20 30 15 20 10 Ctns-/- BMC 10 5 0 0 WT BMC 2 4 2 4 Eye Brain 2.0 70 1.8 60 1.6 1.4 50 1.2 40 1.0 30 0.8 0.6 20 0.4 10 0.2 0.0 0 2 4 2 4
  8. 8. Cystine Content 4 months post-transplant UCSD Biochemical Genetics laboratory Effect of BMC and HSC 250 87% 200 nmol half cystine/mg protein 70% WT Ctns-/- BMC 150 WT BMC 100 82% 94% * 70% 50 * 66% * * * 57% * 0 * Spleen Kidney Brain Heart Eye Liver Muscle *p<0.05
  9. 9. Long-term studies of BMC transplantation (Preliminary data) 15 months-old mice treated for 7 months  Eye: measure of the intraocular pressure 35 30 25 20 WT * * 15 Ctns-/- 10 5 *p<0.05 0 left eye Right eye
  10. 10. Long-term studies of BMC transplantation (Preliminary data) 15 months-old mice treated for 7 months  CNS: Behavioral analyses  Open field Ambulation and center activity normal for Ctns-/- treated and untreated Rearing 80.00 70.00 Vertical counts 60.00 WT untreated 50.00 40.00 CTNS -/- untreated 30.00 20.00 CTNS -/- treated 10.00 0.00 1 2 3 4 5 6 7 8 9 10 11 12 Time interval (5 min)
  11. 11. Long-term studies of BMC transplantation (Preliminary data) 15 months-old mice treated for 7 months  Bone analysis: dual-energy x-ray (DXA) using a PIXImus™ Bone Mineral Content Bone Mineral Density 0.5000 0.0550 0.4800 0.0540 0.4600 0.0530 gram g/cm2 0.4400 * 0.0520 * 0.4200 0.0510 0.4000 0.0500 0.3800 0.0490 WT Ctns-/- Treated Ctns-/- WT Ctns-/- Treated Ctns-/- *p<0.05
  12. 12. Long-term studies of BMC transplantation (Preliminary data) 15 months-old mice treated for 7 months  Kidney analysis cystine content in function of engraftment Serum creatinine in function of engraftment (nmol half-cystine/mg protein) 300.00 0.90 Serum creatinine (mg/dL) 0.80 250.00 0.70 Cystine content 200.00 0.60 0.50 150.00 0.40 100.00 0.30 0.20 50.00 0.10 0.00 0.00 0 20 40 60 80 100 0 20 40 60 80 100 % engraftment % engraftment
  13. 13. Summary 4 months studies  BMC and HSC  Integration in all organs tested: kidney, liver, spleen, heart, muscle, brain and eye  Significant decrease of cystine content in all organs tested  Cell integration increases and cystine content decreases with time Long-term studies  Intro-ocular pressure improved in treated mice  Neuro-motor coordination improved in treated mice  Bone mineral density improved in treated mice  Kidney preservation depends on the BMC engraftment measured in the blood
  14. 14. Relevance This work is a proof of concept for autologous BMC or HSC transplantation for the treatment of cystinosis  Advantages:  currently done in children  no rejection  HSC easily mobilized from peripheral blood  may allow non-myeloablative transplantation  Disadvantage:  in vitro transduction of the patient’s HSC to introduce CTNS gene Need viral vector efficient enough to transduce HSC Need viral vector safe enough to be used Adeno-associated virus (self complementary AAV serotype 1) from Dr. Arun Srivastava (University of Florida)
  15. 15. Next steps  Optimizing HSC transduction and test efficiency and safety of HSC transduced by scAAV1-CTNS on the mouse model for cystinosis  Proof of concept for non-myeloablative HSC/AAV- CTNS transplantation in the mouse model  Non-myeloablative HSC/AAV-CTNS transplantation in Rhesus macaque monkeys  safety  Clinical trial
  16. 16. Acknowledgments Frank Harrison Kimberly Syres, MD Matthew Tadlock, MD Daniel R. Salomon, MD Funding UC Irvine UCSD Biochemical Genetics Lab James V. Jester, MD Jon Gangoiti, MSC Bernadette Libatique UCSD Jerry Schneider, MD Subhojit Roy, MD, PhD Bruce Barshop, MD, PhD Inserm U574 Corinne Antignac, MD, PhD Marie-Claire Gubler, MD, PhD Jeff and Nancy Stack

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