Compacted DNA Nanoparticles for Gene Therapy

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Compacted DNA Nanoparticles for Gene Therapy

• Mark J. Cooper, M.D.

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Barriers to Successful Gene Therapy
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• Survive transport through extracellular space
• Cell uptake
• Resistance to nuclease degradation
• Nuclear transport
• Small size permits entry through nuclear pores
  (25 nm diameter)
• Uncoating to release biologically active nucleic
  acid

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Why Gene Therapy Has Not Worked

• VIRAL VECTORS
• modify a human pathogenic virus to express
  desired gene
• toxic
• unable to be given more than once (innate
  immunity)
• less effective in human tissues than initially
  hoped
• NON-VIRAL VECTORS (before Copernicus)
• toxic in some systems
• generally less effective than viral vectors

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Unimolecular DNA Compaction

• Condensation of a Single Molecule of DNA to Form
  DNA Nanoparticles
• Nuclease resistant stabile in serum
• Rapid cellular and nuclear uptake
• Non-degradative intracellular trafficking
  pathway
• Entry into nucleus of non-dividing cells
• High in vivo transfection efficiency

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DNA NanoparticleComponent Formulation
CK30
PEG maleimide
DNA
CK30PEG10k
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Copernicus Formulation ofPEG-Substituted DNA
Nanoparticles

• PEG-CK30 and plasmid DNA
• lysine counterion determines shape
• single DNA molecule
• reproducible formulation
• homogeneous population
• no aggregation in saline
• DNA ? 12 mg/ml
• nuclease resistant
• stable gt 3 years
• consistent in vivo results
• transfect post-mitotic cells
• PHASE I TRIAL CF

TFA
Acetate
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Stoichiometry of DNA Nanoparticles
8234 bp CFTR expression plasmid CK30PEG10k
16,468 negative charges
MOLECULAR WEIGHT ? 13.1 x 106 gm/mol
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Calculated and Measured Volumes of Compacted DNA
Nanoparticles
J. Biol. Chem. 27832578-32586, 2003
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Characterization Flow Chart of DNA Nanoparticles
TANGENTIAL FLOW FILTRATION
EM A260/A280
removal of excess polycation
EM A260/A280 GEL ANALYSIS TURBIDITY ANALYSIS SALT
STABILITY SERUM STABILITY OSMOLALITY,
pH ENDOTOXIN BIOBURDEN STERILITY
CONCENTRATION DIAFILTRATION
only detectable component is compacted DNA
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Nanoparticle Shape is Determined by Polycation
Counterion at the Time of DNA Mixing

• Trifluoroacetate (TFA)
  ellipsoids
• Acetate
  rods
• Bicarbonate
  rods, toroids
• Chloride
  rods (partial)
• Bromide
  ellipsoids

IMPORTANT FUNCTIONAL CORRELATES FOLLOWING IN VIVO
GENE TRANSFER
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In Vivo Gene TransferNanoparticle Optimization

• Intrapulmonary
• Intradermal
• Intramuscular
• Intravenous
• Topical
• Intracranial
• Intraocular

Polycation composition Polycation
counterion Length of polycation /- PEG PEG
Size Percent PEG substitution /- Targeting
ligands





robust transfection by non-targeted
complexes
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Detection of lacZ After Lung Gene Transfer of DNA
Nanoparticles
Nova Red stain
No primary AB
5/8 mice
3/8 mice
Mol. Ther. 8936-947, 2003
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Compacted DNA NanoparticlesEfficient
Transfection of Post-Mitotic Airway Cells
Intranasal
Intrapulmonary
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Microinjection of Naked DNA or Compacted DNA
Nanoparticles

• EGFP expression plasmid
• Rh-Dextran 155 kD
• live cells imaged
• (no fixation)
• samples blinded before
• analysis

5 kbp plasmid
J. Biol. Chem. 27832578-32586, 2003
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Microinjection of DNA Nanoparticles of Different
Sizes

• GFP Plasmids
• 2.9 kbp
• 5.1 kbp (lambda DNA stuffer fragment)
• 10.7 kbp (lambda DNA stuffer fragment)
• 28 kbp (Mareks virus DNA stuffer fragment)
• Equivalent serum stabilities

J. Biol. Chem. 27832578-32586, 2003
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Size of Compacted DNA Nanoparticle Determines
Nuclear Entry Following Microinjection

J. Biol. Chem. 27832578-32586, 2003
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Effect of DNA Nanoparticle Size on Intrapulmonary
Gene Delivery
Rods
Luciferase Plasmids (kbp) 5.3 9.7 20.2
l DNA stuffer fragments
Ellipsoids
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Plasmid Size and ShapeIntrapulmonary Gene
Transfer
TFA Ellipsoids
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DNA Nanoparticles
What is the mechanism for cell entry?
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In Vitro Transfection of Primary Human Tracheal
Epithelial Cells
15 min
30 min
60 min
DNA
Nuclei
Merged Image
Collaborative studies with Drs. Diane Kube and
Pam Davis, CWRU
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Uptake and Trafficking ofNon-Targeted DNA
Nanoparticles
PRIMARY AIRWAY CELLS
nucleolus
nuclear pore
FITC -- nucleolin Rh -- DNA
NON-DEGRADATIVE TRAFFICKING PATHWAY little
colocalization with antibodies to Rab 5, EEA-1,
cathepsin D, or LAMP-1
nucleolin
cell surface
BINDING TO CELL SURFACE NUCLEOLIN COMPLEX

-
98 0
post-translational modification
DNA nanoparticle
DNA
nucleolin
In collaboration with D. Kube and P. Davis, CWRU
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Tissues with Cell Surface Nucleolin

• Differentiated lung cells (Pam Davis, CWRU)
• Neuronal cells (brain, eye)
• Neovasculature of tumors (diabetic retinopathy?,
  macular degeneration?)
• Tumor cells
• Initiate clinical trial for a pulmonary indication

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Clinical Trial -- Cystic Fibrosis

• The CFTR gene encodes a membrane-bound chloride
  channel mutations in CFTR cause abnormally
  thick, sticky mucus in the lungs that leads to
  recurrent lung infections
• Symptomatic -- no current therapy addresses the
  underlying cause of CF
• 70,000 patients in the US and Europe
• More than 10 million Americans are asymptomatic
  carriers of a defective CFTR gene
• Most patients succumb to progressive respiratory
  failure, with a median age of survival of 33.4
  years

Monogenetic
Treatment
Prevalence
Carriers
Mortality
Source Cystic Fibrosis Foundation
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Correction of Chloride Channel Defect in CFTR -/-
Mice
Mouse 3
Mouse 1
Pre-treatment
Pre-treatment
3 Days post rx
3 Days post rx
NPD
12.5
mV
200 sec
Saline treated
CFTR IHC
Mouse 1
Mouse 3
collaborative studies with A. Ziady and P. Davis
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Single Dose Escalation Study to Evaluate Safety
of Nasal Administration of CFTR001 Gene Transfer
Vector to Subjects with Cystic Fibrosis

• Konstan MW,1 Wagener JS,2 Hilliard KA,1 Kowalczyk
  TH3, Hyatt SL,3 Fink TL,3 Gedeon CR,3 Oette SM3,
  Payne JM3, Muhammad O3, Klepcyk P,3 Peischl A,3
  Davis PB1, Moen RC3, Cooper MJ3

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Department of Pediatrics, Case Western Reserve
University School of Medicine, Cleveland, OH
1 Department of Pediatrics, University of
Colorado Health Sciences Center, Denver, CO 2
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CF Clinical Trial CTI02001

• Phase I
• Placebo-controlled, double-blind
• IN application
• Single dose, dose escalation
• 3 dose levels (12 patients)
• 1/2 log increments (10-fold range)
• 0.40 mg/ml x 2 ml 0.80 mg (2 patients)
• 1.33 mg/ml x 2 ml 2.67 mg (4 patients)
• 4.00 mg/ml x 2 ml 8.00 mg (6 patients)

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CFTR Expression Plasmid
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CF Clinical Trial CTI02001Toxicity Measurements

• Standard clinical/laboratory assessments
• Nasal washings
• baseline
• days 2, 3, 4, 7, 14
• cell count
• cytokines (IL-6, IL-8)
• total protein
• Serum IL-6
• PFTs, CXR, SaO2, CH50, CRP

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CF Clinical Trial CTI02001Toxicity Measurements

• No reportable adverse events
• No adverse events attributed to clinical trial
  material
• (1) grade II pulmonary CF exacerbation at day 14
• all other adverse events grade I
• Mild, transient serum IL-6 rise in 1 subject
• No significant nasal washing IL-6 or IL-8
  elevations
• No other laboratory or test abnormalities
• Data reviewed by DSMB of CFFTI

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CF Clinical Trial CTI02001Efficacy Measurements

• Nasal potential difference measurements
• baseline
• days 2, 3, 4, 7, 14 (or longer if at day 14)
• Nasal cell scrapings
• days 4, 14
• vector DNA (PCR)
• vector CFTR mRNA (RT-PCR)
• endogenous CFTR and GAPDH (RT-PCR)

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Vector DNA Transfection ofNasal Epithelial Cells
Day 14 (both nostrils)
D3 D14 6/6
3/6 6/6 6/6
low high
DOSE LEVEL
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Nasal Potential Difference Analysis

• SOP from CFF Therapeutics Development Network
• Tracings scrambled and read by impartial observer
• Data finalized before code broken
• 7/126 tracings scored as invalid
• catheter movement, excessive signal to noise ratio

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Nasal Potential Difference MeasurementsNormal
and CF Baseline Values
CF Iso response no values lt -5 mV
Normal Iso response mean SD -9.6 5.1 95 CI
-11.0 to -8.2
Standaert, TA et al. Pediatr Pulmonol 37385-392,
2004
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Nasal Potential Difference Correction
Pre-treatment
Day 3
Amiloride
Amiloride
mV
mV
ATP
Zero Cl
ATP
Iso
Zero Cl
Iso
-9 mV
0 mV
0 3 6
9
0 3 6
9
time (min)
time (min)
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Nasal Potential Difference Measurements by Dose
Level
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NPD Corrections
normal
95 CI
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CF Development Plan

• Aerosol development of compacted DNA
• Promoter refinement of payload plasmid
• Repetitive dosing studies
• Surrogate assay development for CFTR chloride
  channel (suitable for intrapulmonary trial)
• Currently dosing intubated rabbits in
  IND-directed efficacy, toxicology, and DNA
  biodistribution studies
• Phase I intrapulmonary aerosol trial Q405

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Compacted DNA Aerosols
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DNA Nanoparticle AerosolsRetain Structural and
Functional Integrity
before
after
MMAD 4.0 microns /- 2.1
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Plasmid Promoter Evaluation in Mice Following
Intrapulmonary Administration


plt0.001
Collaborative studies with Dr. Deborah Gill and
colleagues, University of Oxford
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Maintenance of Biologic Activity After Repeat
Intrapulmonary Dose Administration
Dosing Protocol
Group 4 saline x4
Group 3 saline x3, luc x1
Group 1 CFTR x3, luc x1
Group 2 saline x1, CFTR x2, luc x!
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Pipeline Clinical Indications forNon-Targeted
Nanoparticles

• Intrinsic Lung Disease
• cystic fibrosis
• asthma, COPD, a1-AT deficiency, etc.
• Lung as Bioreactor
• hemophilia A and B
• cancer (anti-metastases peptides), etc.
• Viral Lung Infections (influenza model)
• Parkinsons disease
• Ophthalmology
• Cancer Neovasculature
• Payloads DNA, RNA, siRNA

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Compacted DNA Nanoparticles

• Highly efficient in vivo gene transfer
  (post-mitotic cells)
• up to 20-fold more efficient than any viral
  vector in some tissues
• Efficient and reproducible formulation
  manufactured with readily available cGMP raw
  materials
• Stable gt3 years 4oC 9 months RT
• Non-toxic and non-immunogenic repeat dosing is
  possible
• Encouraging results in human CF clinical trial
• NEW CLASS OF THERAPEUTICS WITH ATTRIBUTES OF
  TRADITIONAL PHARMACEUTICALS

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Counterion of PEG-substituted Polycation
Influences Shape of DNA Nanoparticles
Acetate
HCO3
100 nm
100 nm
Chloride
TFA
100 nm
100 nm
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Benign Preclinical Toxicology

• High Dose (30-fold higher than highest dose in
  trial)
• trace to 1 mononuclear cell infiltrate around
  pulmonary veins
• no peribronchial or alveolar infiltrates
• Low Dose (3-fold higher than highest dose in
  trial)
• no histologic findings
• no blood test abnormalities, including complement
• minimal BAL cytokine induction after
  intrapulmonary dose
• no/minimal CpG island response