1. Combination Gene Therapy,
Bone Marrow Transplantation
and Substrate Reduction Therapy
for Globoid Cell Leukodystrophy
(Krabbe Disease)
Mark S. Sands, Ph.D.
Washington University School of Medicine

2. Globoid-Cell Leukodystrophy
- CNS and PNS involvement
- Galactocerebrosidase deficiency (lysosomal enzyme)
- Demyelinating disease
- Rapid psychomotor deterioration
- Seizures
(Krabbe Disease)
- Premature death (~2-5 yr)
-Current treatment strategy
- bone marrow transplantation
- cord blood transplantation
- partially effective

3. Murine Model of Krabbe Disease
(Twitcher Mouse)
• Galactocerebrosidase-deficient
• De/Dysmyelinating disease
• Central and peripheral neuropathy
• Infiltrating immune cells
• Rapid progression (life span ~40d)
• Severe tremor
• Hind limb ataxia
• BMT partially effective
– Day 10 – 80d life span
– Day 1 – 45d life span
WT Twi

4. CNS-Directed AAV-Mediated Gene Therapy
&
Bone Marrow Transplantation

5. Single Therapies-
Too Narrow a Focus?
• GLD is a complex disease involving multiple
systems and mechanisms
Primary Genetic Defect
Inflammation
Death and Destruction
Bone Marrow
Transplant
Gene Therapy
Accumulation of substrates
?
?

6. Combination Therapy
(AAV2/5 and BMT)
Genotype
PND2 PND3 PND4
i.c. and i.t. injection
of GALC-AAV2-5
400rads
radiation
i.v. injection of
GFP+ BM cells

7. GALC Activity
Reddy AS, et al., (2011) J. Neurosci 31:9945

8. Psychosine

9. Behavior, etc

10. Life Span
(≈130 d)
(≈70 d)

11. Mechanism of Synergy

12. Neuroinflammation

13. Astrocytosis (GFAP)

14. Activated microglia/macrophages (CD68)

15. Life Span
(≈130 d)
(≈70 d)

16. CNS-Directed AAV-Mediated Gene Therapy
&
Bone Marrow Transplantation
&
Substrate Reduction Therapy

17. Triple Combination
(Rationale)
Primary Genetic Defect
Inflammation
Death and Destruction
Bone Marrow
Transplant
Gene Therapy
Accumulation of substrates
Substrate
Reduction
Therapy
L-cycloserine
Biswas & Levine (2002)
Pediatric Research 51:40.

18. AAV2/5, BMT and L-cycloserine
experimental design
Genotype
PND2 PND3 PND4
i.c. and i.t. injection
of GALC-AAV2-5
400rads
radiation
i.v. injection of
GFP+ BM cells
3X weekly injections
of L-cycloserine
50mg/kg

19. Conclusions
1) Single treatments are minimally to marginally effective
for Krabbe disease
2) CNS-directed gene therapy and BMT synergize to
greatly increase efficacy
3) BMT provides immunomodulatory effects
4) Further synergy is observed when substrate reduction therapy
is added to gene therapy and BMT.
Question?
1) Can further synergistic improvements be achieved by
treating additional therapeutic targets?

20. Acknowledgements
Sands Lab:
• Shannon Macauley
• Marie Roberts
• Josh Woloszynek
• Adarsh Reddy
• Kevin O’Dell
• Sarah Hohm
• Elizabeth Qin
• Jacqui Hawkins
• Tom Daly
• Darshong Lin
• Anthony Frisella
Support
• NIH - NS43205 & HD55461
• NRSA Fellowship F31 NS056718
• BDSRA Postdoctoral Fellowship
• Hunter’s Hope
• Legacy of Angels Foundation
• Tay-Sachs and Allied Diseases
King’s College - London
• Catherine Kielar
• Andrew Wong
• Jonathon Cooper
Washington University:
• Mike Wong (Seizure Monitoring)
• Mingjie Li (Viral Vector Core)
• Jin-Moo Lee (GFAP, Vim mice)
• Ernie Gonzales (Animal surgery Core)
UI Chicago
• Ernesto Bongarzone