therapeutic potential of designer receptors, gary aston-jones, phd
TRANSCRIPT
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Guan et al, Intl J Obesity, 2002.
Different types of neurons are interdigitated in the same brain areas:
How selectively stimulate/inhibit?
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Davidson & Breakefield, Nat. Rev. Neurosci. 2003.
Gene transfer in brain in vivo
viral vectors
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Calloway, E., Trends Neurosci. 2005.
Cell-type-specific gene transfer & modulation of
neuronal activity
Allostatin (AL) = invertebrate peptide/receptor system
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Evolving the lock to fit the key: Designer
receptors exclusively activated by designer
drugs (DREADDS)
Conklin, B., PNAS (2007)
Armbruster et al., PNAS (2007)
directed evolution: muscarinic ACh receptor -> DREADD
CNO=clozapine n-oxide
Muscarinic Ach receptor
DEADD
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Courtesy of Bryan Roth, U North Carolina
Specific neuronal inhibition with hM4D DREADD
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Lentivirus to drive DREADD via orexin promoter: selective activation or inhibition of orexin
neurons in behaving animals
WPRE
ampR
HIV Sequence
HIV LTR'
hORX p
EGFP
LTRPsi
IRES
DREADD
SOWVG-D13880 bp
•Brain orexin/hypocxretin system
• Arousal, stress• Reward, addiction• Eating, obesity
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Photosensitive ion channels for control of neuronal activity
Zhang et al., Nature Rev. Neurosci. (2007)
Channelrhodopsin Halorhodopsin
excitation inhibition
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Photostimulation yields more specific effects than electrical stimulation
Alternative to Deep Brain Stimulation
Zhang et al., Nature Rev. Neurosci. (2007)
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Cell-type specific expression of channelrhodopsin in brain norepinephrine neurons in vivo
PRSx8 promoter; mCherry tagMicroinjection in vivo in locus coeruleus
TH (NE neurons) mCherry (ChR2 neurons) Merge
David MoormanElena Vazey
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Photostimulation of CNS neurons in vivo
record laser
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Photostimulation of neuronal activity in vivo
Locus coeruleus neuronal recordings in anesthetized rat
David MoormanElena Vazey
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Summary
• Novel proteins designed and expressed in neurons• Designer receptors• Photosensitive channels/ion pumps
• Neurons transduced in vivo to express novel proteins• Viral vectors (lenti virus, adeno-associated virus, etc)• Cell-type specific expression using promoters
• Many clinical applications• Designer receptors (DREADDS): More specific than conventional pharmacology• Photostimulation: More specific, less toxic than Deep Brain Stimulation (DBS)• Much better targeting ability than conventional approaches, e.g., DBS, lesion• Many fewer side-effects
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Acknowledgements
Rachel SmithDavid MoormanAngie CasonPouya Tashili-FahadanKimberlei RichardsonSteve MahlerLana ZhangGreg SartorElena VazeyMike SmithBecky FallonGlenda Harris (U Penn) Matt Wimmer (U Penn)
MUSC Neuroscience of Addiction Research Center
National Institute on Drug AbuseLilly Pharmaceuticals
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1414
Transfer Vector DesignTransfer Vector Design
PromotersPromoters– mPGK-1mPGK-1– hSyn-1: neuronshSyn-1: neurons– hGFAP: gliahGFAP: glia– CaM kinase IICaM kinase II– rMORrMOR– rPPErPPE
Internal ribosome entry sites (IRES)Internal ribosome entry sites (IRES)– Encephalomyocarditis virus (ECMV)Encephalomyocarditis virus (ECMV)– Synthetic (Gtx)Synthetic (Gtx)
Woodchuck hepatitis virus post-translational Woodchuck hepatitis virus post-translational regulatory element (WPRE)regulatory element (WPRE)
Promoter Transgene IRES EGFP WPRE