1. Article: "Noninvasive and Targeted Gene Delivery into
the Brain Using Microbubble-Facilitated Focused
Ultrasound"
Malova Anna
06.04.2013
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2. Introduction
rAAV
Gene therapy is a potentially powerful means of treatment of various diseases with
genomic causes. Recombinant adeno-associated viral (rAAV) vectors
No pathogenicity
Typical persistence of the transgene
Low immunogenicity
Complete removal of all viral genes
Long-term gene expression
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3. Introduction
Using
AAV serotype 2 (AAV2) vectors uses for the treatment of various CNS diseases as
Canavan’s disease
Batten’s disease
Parkinson’s disease
Alzheimer’s disease
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4. Introduction
Blood-Brain Barrier
Separation of circulating blood from the brain extracellular fluid in the central
nervous system
Protects the brain tissue from circulating microorganisms and toxins
Actively transport metabolic products such as glucose across the barrier with
specific proteins
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5. Introduction
Problems
Existence of the blood-brain barrier (BBB)
Intravenous injection is ineffective
Need local, direct injection into the brain
Region of recombinant gene-expression is severely limited
Transfected cells cannot be widely spread
Additional risks
Burr holes
Specific targeted gene expression cannot be controlled
Using IV injection other organs would be significantly infected
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6. Introduction
Microbubble-enhanced focused ultrasound (FUS)
Locally and temporally disrupt the BBB
Noninvasive method
Contrast-enhanced magnetic resonance imaging can be used to observe,
monitor and guide
Low viral titer of 1 × 109
viral genomes
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7. Goal
Experimental scheme
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8. Goal
Preparing
Outbred mice were separated into two groups
Group 1 were used to optimize the time course of AAV infection from 1 to 6
weeks
Group 2 used to evaluate the efficiency of approach at the optimized time for
infection of 2–3 weeks.
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9. Goal
Experiment
Catheter was inserted into the tail vein for injections
Viral vectors with the titer of 1 × 109
viral genomes per gramm were bolus
injected through catheter
Immediately followed a microbubbles bolus injection
Ultrasonic energy was delivered to the brain transcranially using a spherically
focused transducer
Animals were sacrificed after FUS sonication. The brains of these mice were
quickly removed and frozen
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10. Results
GFP Expression
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11. Results
GFP Expression. Explanation
Determine the kinetics of infection in the BBB-opened brain region (top)
In the absence of BBB-opening, GFP expression did not detect
GFP expression could be observed within the first week
Reached a maximum level at week 3, after which it decayed.
AAV was only capable of transducing cells located in the vicinity of the
BBB-breakdown injection track region but could not infect the parenchyma
containing an intact BBB structure
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12. Results
Correlation between MRI and GFP expressions
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13. Results
Correlation between MRI and GFP expressions. Explanation
GFP expression sites colocalized well with the contrast-enhanced regions
observed in MRT images from the same animals
High correlation between GFP expression and MRI signal increase
Higher degree of BBB-opening induced a greater level of AAV transfection
and expression
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14. Discussion
Discussion
Unlike current invasive procedures involving direct local injection of viral
vector, FUS procedure concentrated ultrasound transcranially to induce local
BBB opening for expressing genes at specific target regions in a noninvasive
manner.
GFP expression correlated well with MRI signal enhancement, suggesting the
possibility of using MRI-monitored BBB-opening
Since viral transduction of specific cells relies on the promoters that drive
viral vectors, it is important to select promoters that will result in efficient
gene expression in the selected target cells
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15. Discussion
Future Directon
The given viral vector in this study should be of no safety concern, and there
should be highly possible to further improve the gene expression rate and
transduction distribution when higher titer of viral vector is employed
Combined use of viral-vector intravenous administration with FUS-BBB
opening is a potential technique to achieve targeted gene delivery for CNS
disease treatment noninvasively.
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16. Questions
Questions
Thank you for your attentions! Questions?
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