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

This work is licensed under the Creative Commons Attribution 3.0 United States License. To view a copy of this license, visit:http://creativecommons.org/licenses/by/3.0/us/ or send a letter to Creative Commons, 171 Second Street, Suite 300, San Francisco, California, 94105, USA.

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