Compacted DNA Nanoparticles for
Gene Therapy
Mark J. Cooper, M.D.
Copernicus Therapeutics, Inc.
Barriers to Successful Gene Therapy
1
1. Survive transport through
extracellular space
2
2. Cell uptake
3
3. Resistance to nuclease degradation
6
4. Nuclear transport
4
5
6
5. Small size permits entry through
nuclear pores (25 nm diameter)
6. Uncoating to release biologically
active nucleic acid
Why Gene Therapy Has Not Worked
z
VIRAL VECTORS
„
„
„
„
z
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
Unimolecular DNA Compaction
Condensation of a Single Molecule of DNA to
Form DNA Nanoparticles
z
z
z
z
É
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
DNA Nanoparticle:
Component Formulation
CK30
DNA
PEG maleimide
CK30PEG10k
Copernicus Formulation of
PEG-Substituted DNA Nanoparticles
PEG-CK30 and plasmid DNA
z lysine counterion determines shape
z single DNA molecule
z reproducible formulation
z homogeneous population
z no aggregation in saline
z [DNA] > 12 mg/ml
z nuclease resistant
z stable > 3 years
z consistent in vivo results
z transfect post-mitotic cells
z PHASE I TRIAL CF
z
100 nm
TFA
100 nm
Acetate
Stoichiometry of DNA Nanoparticles
8234 bp CFTR expression plasmid + CK30PEG10k
16,468 negative charges
DNA
CK30PEG10k
MOLES PER COMPLEX
1
549
Bound Charge Ratio
1.5
2.9 kbp
1.0
1.0
5.1 kbp
28 kbp
0.5
0.0
0.0
MOLECULAR WEIGHT ≅ 13.1 x 106 gm/mol
1.5
0.5
0.5
1.0
1.5
2.0
2.5
3.0
0.0
3.5
Input Charge Ratio
J. Biol. Chem. 278:32578-32586, 2003
Calculated and Measured Volumes of
Compacted DNA Nanoparticles
A1 H1
50
2.9 kbp
Frequency
40
A2 H2
30
20
10
0
0
10000
20000
30000
40000
50000
Volume (nm3)
J. Biol. Chem. 278:32578-32586, 2003
Characterization Flow Chart of DNA
Nanoparticles
EM
A260/A280
EM
A260/A280
GEL ANALYSIS
TURBIDITY ANALYSIS
SALT STABILITY
SERUM STABILITY
OSMOLALITY, pH
ENDOTOXIN
BIOBURDEN
STERILITY
TANGENTIAL
FLOW FILTRATION
removal of
excess polycation
CONCENTRATION
DIAFILTRATION
only detectable component
is compacted DNA
Nanoparticle Shape is Determined by Polycation
Counterion at the Time of DNA Mixing
Trifluoroacetate (TFA)
z Acetate
z Bicarbonate
z Chloride
z Bromide
z
ellipsoids
rods
rods, toroids
rods (partial)
ellipsoids
IMPORTANT FUNCTIONAL CORRELATES
FOLLOWING IN VIVO GENE TRANSFER
In Vivo Gene Transfer
Nanoparticle Optimization
** z Intrapulmonary
** z Intradermal
* z Intramuscular
Intravenous
z Topical
** z Intracranial
*** z Intraocular
z
Polycation composition
Polycation counterion
Length of polycation
+/- PEG
PEG Size
Percent PEG substitution
+/- Targeting ligands
** robust transfection by “non-targeted” complexes
Detection of lacZ After Lung Gene
Transfer of DNA Nanoparticles
Nova Red stain
No primary AB
5/8
mice
3/8
mice
Mol. Ther. 8:936-947, 2003
Compacted DNA Nanoparticles:
Efficient Transfection of Post-Mitotic Airway Cells
A
Intranasal
Intrapulmonary
B
Microinjection of Naked DNA or
Compacted DNA Nanoparticles
100
Naked Nucleus
z
EGFP expression plasmid
50
Compacted Nucleus
z
Rh-Dextran 155 kD
25
Naked Cytoplasm
z
live cells imaged
Green cells %
75
***
Compacted Cytoplasm
20
(no fixation)
***
***
10
z
*
0
0.1
1.0
10.0
100.0
1000.0
5 kbp plasmid
[DNA] (μg/ml)
J. Biol. Chem. 278:32578-32586, 2003
samples blinded before
analysis
Microinjection of DNA Nanoparticles of
Different Sizes
50
GFP Plasmids
2.9 kbp
„ 5.1 kbp (lambda DNA stuffer fragment)
„ 10.7 kbp (lambda DNA stuffer fragment)
„ 28 kbp (Marek’s virus DNA stuffer fragment)
„
z
28
40
Minor length (nm)
z
30
5.1
10.7
2.9
20
Equivalent serum stabilities
10
10
20
30
40
50
60
70 80 90 100
Major length (nm)
J. Biol. Chem. 278:32578-32586, 2003
Size of Compacted DNA Nanoparticle Determines
Nuclear Entry Following Microinjection
EGFP Positive Cells (%)
60
Nucleus, Compacted DNA
50
Nucleus, Naked DNAl
40
Cytoplasm, Compacted DNA
30
15
Cytoplasm, Naked DNA
**
10
5
0
15
20
25
30
35
40
Minor length (nm)
45
50
J. Biol. Chem. 278:32578-32586, 2003
Effect of DNA Nanoparticle Size on
Intrapulmonary Gene Delivery
Rods
Luciferase Plasmids (kbp)
λ DNA
stuffer
fragments
5.3
9.7
20.2
Ellipsoids
10
10 1
Major Diameter (nm)
TFA 20.2 luc
TFA 9.7 luc
20
TFA 5.3 luc
30
Ac 20.2 luc
40
Ac 9.7 luc
50
log (RLU/mg
protein/pmol DNA
above background)
TFA Ellipsoids
Ac 5.3 luc
60
70
80
90
100
50
40
30
20
10
Minor Diameter (nm)
Plasmid Size and Shape:
Intrapulmonary Gene Transfer
10 6
10 5
10 4
10 3
10 2
DNA Nanoparticles
What is the mechanism for cell entry?
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
Uptake and Trafficking of
Non-Targeted DNA Nanoparticles
PRIMARY
AIRWAY CELLS
nucleolus
nuclear
FITC -- nucleolin
Rh -- DNA
pore
NON-DEGRADATIVE
TRAFFICKING PATHWAY
little colocalization with
antibodies to Rab 5, EEA-1,
cathepsin D, or LAMP-1
cell surface nucleolin
+
BINDING TO CELL
SURFACE NUCLEOLIN
COMPLEX
-
98%
0%
DNA+
post-translational
modification
In collaboration with D. Kube and P. Davis, CWRU
DNA nanoparticle
nucleolin
Tissues with Cell Surface Nucleolin
Differentiated lung cells (Pam Davis, CWRU)
z Neuronal cells (brain, eye)
z Neovasculature of tumors (diabetic retinopathy?,
macular degeneration?)
z Tumor cells
z
É
Initiate clinical trial for a pulmonary indication
Clinical Trial -- Cystic Fibrosis
Monogenetic
z
Treatment
z
Prevalence
z
Carriers
z
Mortality
z
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
Source: Cystic Fibrosis Foundation
Correction of Chloride Channel Defect in
CFTR -/- Mice
Mouse 3
Pre-treatment
NPD
Mouse 1
3 Days post rx
Pre-treatment 3 Days post rx
12.5
mV
200 sec
Saline treated
CFTR
IHC
Mouse 3
Mouse 1
collaborative studies with A. Ziady and P. Davis
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
Copernicus Therapeutics, Inc.
3
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
CF Clinical Trial CTI02001
Phase I
z Placebo-controlled, double-blind
z IN application
z Single dose, dose escalation
z 3 dose levels (12 patients)
z
„ 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)
CFTR Expression Plasmid
KmR
CMV Enhancer
CMV promoter
ColE1 origin
CMV intron A
HTLV-Ru5
BGH polyA
pKCPIRCFTRBGH(-)
8234 bp
hCFTR
CF Clinical Trial CTI02001:
Toxicity Measurements
Standard clinical/laboratory assessments
z Nasal washings
z
„ baseline
„ days
2, 3, 4, 7, 14
– cell count
– cytokines (IL-6, IL-8)
– total protein
Serum IL-6
z PFTs, CXR, SaO2, CH50, CRP
z
CF Clinical Trial CTI02001:
Toxicity Measurements
No reportable adverse events
z No adverse events attributed to clinical trial material
z
„ (1)
grade II pulmonary CF exacerbation at day 14
„ all other adverse events grade I
Mild, transient serum IL-6 rise in 1 subject
z No significant nasal washing IL-6 or IL-8 elevations
z No other laboratory or test abnormalities
z Data reviewed by DSMB of CFFTI
z
CF Clinical Trial CTI02001:
Efficacy Measurements
z
Nasal potential difference measurements
„ baseline
„ days
z
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)
Vector DNA Transfection of
Nasal Epithelial Cells
6/6
3/6
6/6
0.01
0
DNA DLIII
high
6/6
0.1
SALINE DLIII
low
D14
1
DNA DLI/II
D3
TOTAL
10
SALINE DLI/II
DOSE LEVEL
NUMBER OF
SUBJECTS +
DNA COPY NUMBER/CELL
Day 14 (both nostrils)
Nasal Potential Difference Analysis
z
SOP from CFF Therapeutics Development Network
z
Tracings scrambled and read by impartial observer
z
Data finalized before code broken
z
7/126 tracings scored as invalid
„
catheter movement, excessive signal to noise ratio
Nasal Potential Difference Measurements
Normal and CF Baseline Values
CF Iso response
no values < -5 mV
Normal Iso response
mean ± SD = -9.6 ± 5.1
95% CI = -11.0 to -8.2
Standaert, TA et al. Pediatr Pulmonol 37:385-392, 2004
Nasal Potential Difference Correction
Pre-treatment
Day 3
Amiloride
mV
mV
Amiloride
Zero Cl
ATP
Iso
Zero Cl
ATP
Iso
-9 mV
0 mV
1 min
3
time (min)
6
9
0
10 mV
10 mV
0
1 min
3
time (min)
6
9
Nasal Potential Difference Measurements
by Dose Level
Number of Subjects
4
≥ -5 mvolts
< -5 mvolts
3
2
1
0
DL I
DL II
DL III
NPD Corrections
Data from Both Nostrils
normal
95% CI
delta NPD(iso) (mV)
5
0
-5
-10
-15
-30
Baseline
mean
Days 1-7
Day 13
001
002
003
004
005
006
007
008
009
010
011
012
CF Development Plan
z
z
z
z
z
É
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
Compacted DNA Aerosols
% of Total Recovered DNA
Battelle Study N104881
Cascade Impactor Filters
35
Alveoli Proximal
Airw ay
30
25
20
15
10
5
0
<0.6 1 2 3 4 5 6 7 8 9 10 11 12 >12
Mean Droplet Size (μm)
Aeroneb Run # 1
Aeroneb Run # 2
Pari Run # 1
Pari Run # 2
DNA Nanoparticle Aerosols
Retain Structural and Functional Integrity
HepG2 transfection with compacted luciferase DNA
before or after aerosolization
RLU/mg protein
10000000
before
1000000
100000
10000
1000
before
10ng
1ng
10ng
100 nm
1ng
mock
100
after
PD-IN072503
Luciferase Data, Day 2 Collection
after
100 nm
MMAD 4.0 microns +/- 2.1
RLU/mg protein
10 6
10 5
10 4
10 3
10 2
saline
before
after
Plasmid Promoter Evaluation in Mice
Following Intrapulmonary Administration
Geometric Means of
RLU/mg protein (±SEM)
PD-IN102402
10
5
RT285 CMV
RT277 pUb
Saline
***
***
10 4
10 3
10 2
2
7
14
28
Collection Day
35
*** p<0.001
Collaborative studies with Dr. Deborah Gill and colleagues, University of Oxford
Maintenance of Biologic Activity After Repeat
Intrapulmonary Dose Administration
Dosing Protocol
PD-IN120503 Repeat Dosing #2
Luciferase Data
10 7
3 weeks
3 weeks
saline
CFTR
saline
saline
luc
CFTR
3 weeks
CFTR
luc
Group 2.
saline
luc
Group 3.
saline
saline
saline
10 6
2d
saline
Group 4.
RLU/mg protein
CFTR
Group 1.
CFTR
10 5
10 4
10 3
10 2
10 1
10 0
harvest
p4 4
u
o x
Gr line
sa
p1 1
p2 !
p3 1
u
u
u
x
o x
o
o x
Gr luc
Gr , luc
Gr luc
,
,
x2
x3
x3
R
e
R
T
in
FT
sal
CF
C
,
x1
e
n
i
sal
Pipeline Clinical Indications for
“Non-Targeted” Nanoparticles
z
z
z
z
z
z
z
Intrinsic Lung Disease
„ cystic fibrosis
„ asthma, COPD, α1-AT deficiency, etc.
Lung as Bioreactor
„ hemophilia A and B
„ cancer (anti-metastases peptides), etc.
Viral Lung Infections (influenza model)
Parkinson’s disease
Ophthalmology
Cancer Neovasculature
Payloads: DNA, RNA, siRNA
Compacted DNA Nanoparticles
z
Highly efficient in vivo gene transfer (post-mitotic cells)
„
z
z
z
z
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 >3 years 4oC; 9 months RT
Non-toxic and non-immunogenic; repeat dosing is possible
Encouraging results in human CF clinical trial
É
NEW CLASS OF THERAPEUTICS WITH
ATTRIBUTES OF TRADITIONAL
PHARMACEUTICALS
Counterion of PEG-substituted Polycation
Influences Shape of DNA Nanoparticles
HCO3
Acetate
100 nm
100 nm
Chloride
TFA
100 nm
100 nm
Benign Preclinical Toxicology
z
High Dose (30-fold higher than highest dose in trial)
„
„
z
Low Dose (3-fold higher than highest dose in trial)
„
z
z
trace to 1+ mononuclear cell infiltrate around pulmonary veins
no peribronchial or alveolar infiltrates
no histologic findings
no blood test abnormalities, including complement
minimal BAL cytokine induction after intrapulmonary dose
„
no/minimal CpG island response