Gene Therapy - Welcome to INTERNAF
Transcript of Gene Therapy - Welcome to INTERNAF
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Neural Stem Cells NeurodegenerativeDisorders
Gene Therapy
Schaffer LabChemical Engineering
U.C. Berkeley
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Neuronal Protection and Replacement
Gene therapy for neuroprotection:ALS and spinocerebellar ataxia
Gene delivery for control of adultstem cells
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Neuronal Protection and Replacement
Gene delivery for neuroprotection:ALS and spinocerebellar ataxia
Gene delivery for control of adultstem cells
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Stem Cell Therapy: In Vitro & In Vivo
Harvest, Grow, andReimplant
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Stem Cell Therapy: In Vitro & In Vivo
Direct Control in theNervous System by
Delivery of Drugs orGenes
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Underlying Challenge: How to Control theStem Cells
?
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Underlying Challenge: Cellular Control
Signals(t)
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Adult Neural Stem Cells
Shown very recently to exist throughout the nervoussystem
Significant potential for neural regeneration
Requires better understanding of signaling mechanismsthat regulate these cells’ function
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1) Site of active adult neural stem cells2) Site affected by Alzheimer’s Disease
Brain Section: Hippocampus
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Propagation of Immature Neural Stem Cells
Blue - nucleus
Green - nestin
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Differentiation of Stem Cells into Glia
Blue - nucleus
Green - MBP
Red - GFAP
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Differentiation into Neurons
Blue - nucleus
Green - NF200
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Goal: Control Neural Stem Cell Behavior
Stem Cell ProgenitorCell
Glia
Neurons
?
?
?
?
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Goal: Control Neural Stem Cell Behavior
Stem Cell ProgenitorCell
Glia
Neurons
?
?
Sonic Hedgehog
?
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Blue - nucleus
Green - nestin
Red - Shh receptor (Ptc)
Adult Neural Stem Cells Express Ptc
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-0.200
0.300
0.800
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2.300
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0 4 6 8
Day
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Shh Stimulates Neural Progenitor Proliferation
nM
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Adeno-associated Viral Gene Delivery Vehicles
Advantages:
+ Extremely safe
+ Highly efficient
+ Very stable expression
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Adeno-associated Viral Gene Delivery Vehicles
Green FluorescentProtein
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AAV-GFP Delivery to the Brain
after 1 year
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Adeno-associated Viral Gene Delivery Vehicles
Sonic Hedgehog
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Green - neurons (NeuN)
Red - mitotic cells (BrdU)
Adult Neural Stem Cell Proliferation: Control
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Adult Neural Stem Cell Proliferation: AAV-Shh
Green - neurons (NeuN)
Red - mitotic cells (BrdU)
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Shh Triples Neural Stem Cell Proliferation
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AAV-GFP AAV-Shh
#BrdU+ cells/section
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Shh Triples New Neurons
#BrdU+ cells/section
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Shh BrdU GFP BrdU ShhBrdU/NeuN
GFPBrdU/NeuN
Lai et al., Nature Neuroscience (2003)
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Neuronal Protection and Replacement
Gene delivery for neuroprotection:ALS and spinocerebellar ataxia
Gene delivery for control of adultstem cells
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Gene Therapy: Concept andCurrent Status
Definition:the delivery of genetic material to an individual’s cells for therapeutic benefit
Recent success:Hemophilia B using AAV (Avigen)Heart disease using adenovirus (Coll.Thx.)Cancer using adenovirus (Onyx)
Challenge:Need better gene delivery technology
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Adeno-associated Viral Gene Delivery Vehicles
Green FluorescentProtein
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AAV Neuroprotection in the Spinal Cord
Fred Gage Lab, Salk InstituteKaspar et al., Science (2003)
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Amyotrophic Lateral Sclerosis
• Progressive disease that selectively kills spinal cordmotor neurons (Lou Gherig’s Disease, Stephen Hawkins)
• Fatal within 1-5 years of onset
• Prevalence of 2-3 per 100,000 people
• Causes of disease remain unknown
• 5-10% of cases, inherited in a dominant manner [SOD-1mutations (Superoxide dismutase-1) 90 mutations known
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AAV Gene Therapy for ALS
NeuroprotectiveFactorIGF-I
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Gene Delivery Protects Motor Neurons
Kaspar et al., Science (2003)
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Gene Therapy Significantly Delays Symptoms
Kaspar et al., Science (2003)
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Neuronal Protection and Replacement
Gene delivery for neuroprotection:ALS and spinocerebellar ataxia 3
Gene delivery for control of adultstem cells
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Challenge for Dominant Disorders:Blocking Defective Genes
RNA interference can degrade mutated RNA sequences
RNA interference
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RNA Interference to Knock Down Mutant Ataxin
Gene delivery can reduce mutant Ataxin expression in cell cultureMiller et al., PNAS (2003)
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Gene delivery can be used to control adult neural stem cells in thenervous system for neuron replacement
Gene therapy can be used to deliver genes for generalneuroprotection
Targeted degradation of defective mRNA
Animal models of ataxia
Combining stem cell therapy and gene therapy may prove apowerful approach
Summary
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Acknowledgments
Adult Neural Stem Cells
Karen LaiBrian KasparFred H. Gage
ALS Work/AAV Studies
Brian KasparJeffrey Rothstein
Fred GageProject ALS
Ataxia
Victor Miller et al.Henry Paulson