understanding and re-engineering nucleoprotein machines to cure human disease william s. dynan...
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Understanding and Re-engineering Nucleoprotein Machines to Cure
Human Disease
William S. Dynan
Medical College of Georgia
Nanomedicine Center for Nucleoprotein Machines
© William S. Dynan 2010 licensed under the Creative Commons Attribution 3.0 United States License
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Theme for today:n Development of simple nanodevices (one or
two components) that interface with complex nucleoprotein machines.
n Our models are machines that repair DNA double-strand breaks
n Three examples:n Bright photostable probes to visualize assemblyn Modified single-chain antibody for inhibitionn Gain of function for gene correction
DSBs: How are they formed and why are they important?
n Ionizing radiation and recombination nucleases are main natural sources
n Particle or photon transfers energy to water or other molecules that it encounters along its track.
n Nanoscale distribution of damage is determined by track structure and “linear energy transfer.”
n Unrepaired/misrepaired breaks are gravely dangerous.
DNA-PKcs
Assembly of the nonhomologous end joining machine
L4/X4/XLF
Ku70/80* *
Chromatin modification – gamma-H2A.XSensors and transducers of DNA damage response - 53BP1
50 nm
Example 1: Bright photostable probes to visualize repair complex assembly
Fluorescent protein Orthogonal tagging QD tagging
Controlled induction: stage-mounted microirradiator
Steeb J, Josowicz M, Janata J, Nickel-63 microirradiatorAnal Chem 81:1976-1981 (2009)
~25 mm beam
Visualization of complex assembly in real time
YFP-53BP1 tandem tudor domain, deconvolution microscopy
Assembly (1 hour timescale) Disassembly (8 hour timescale)
Example 2: modified single chain antibody for repair inhibition
DNA-PKcsKu70/80
~ 1 million radiotherapy patients per year in North AmericaTumor cells lack damage-dependent cell cycle checkpoints
Replicate unrepaired DNA/ dividePost-mitotic cell death
Delayed/absent DSB repair potentially increases therapeutic gain
Inhibitor: modified ScFv 18-2
n Small (30 kDa) is monoclonal antibody derivativen Recognizes a conserved regulatory sequence in the center of DNA-PKcs
(residues 2001 to 2025).
3-4 nm
Macromolecular delivery methods
n Chemical/mechanicaln Microinjection
n Precise volume and timingn Unmodified ligandn Control cells on same plate
n Receptor-mediated endocytosis
n Allows for cell-specific targetingn Well established in vivo delivery
method Li et al., Nucleic Acids Res 31: 5848-57 (2003)
Folate receptor-mediated delivery
scFv 18-2
Folate
FR
Receptor is over-expressed in cancers.
Ligand binding promotes non-destructive internalization and release of cargo
Proven for model proteins
Clinically applicable
Folate conjugation ofMBP-ScFv 18-2
TWO VERSIONSn Folate-scFvn Folate-HA-scFv
(with endosome disruptor peptide)
n Folate detected by UV spectroscopy
n Confirmed by SDS-PAGE
n Minimal interference with epitope recognition
Folate (ligand) S S ScFv 18-2
Folate (ligand) S S ScFv 18-2HA peptide
Radiobiology: sensitization enhancement
Inhibition of autophosphorylation
Radiosensitizer summary
n Radiobiology (sensitization enhancement) is promising
n Further optimization of design/production underway
n Live cell imaging and animal experiments plannedn Establishes a discovery paradigm
Disease Device Delivery
Example 3: Re-engineering for gene correction
n DNA repair is nature’s only way to alter gene sequences
n Core NHEJ acquired a new function 400 million years ago
n Single additional protein component – encoded by Rag1/2 – promotes combinatorial joining of antigen receptors
n Normally requires NHEJ, although mutant Rag proteins can engage HR.
Adaptive immune system:V(D)J
recombination
Example 3: Re-engineering for gene correction
n Disease: sickle cell anemia/hemoglobinopathiesn Accessible stem celln Monogenic, recessiven Common worldwide (90,000 cases in US)n Life shortening/devastating symptom complexn Faithful animal model
n Device: incision/gene conversionn Delivery: receptor-mediated endocytosis
Disease Device Delivery
Concept: gene correction in the hematopoietic stem/progenitor cell
n Zinc-Finger Nucleases create a DSB near the E6V mutation
n Repair pathway engaged - Rad51 forms presynaptic filament at the DSB site
n Rad51 filament initiates HR with a donor template
Progress and challenges
n Devicen ZFNs available for model genes and for globin
n Deliveryn Receptor-mediated endocytosis shows promise for delivery to
hematopoietic stem cells – autologous re-engraftmentn The challenges are efficiency and specificity
n Real-time visualization of reaction steps provides an approach for systemic optimization of efficiency
n Must be able to monitor and suppress mutation and rearrangementsn An ideal gene correction machine would be independent of foreign
DNA/proteins, amenable to temporal control
Acknowledgment
Nanomedicine Center for Nucleoprotein Machines
Gang Bao – Georgia Tech. Bill Dynan – MCG David Roth – NYU, Steffen Meiler – MCG
Matt Porteus - UTSW
Dynan LabNanomedicine Group
Zhen Cao, Shuyi Li, Bill Dynan, Deepika Goyal, Zhentian Li
Re-engineering therepair machine
Inhibition of response
Imaging DSB responseDSB and response