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Copernicus Therapeutics, Inc.
Compacted DNA Nanoparticles for Gene Therapy
Mark J. Cooper, M.D.
1. Survive transport through extracellular space
2. Cell uptake
3. Resistance to nuclease degradation
4. Nuclear transport
5. Small size permits entry through nuclear pores (25 nm diameter)
6. Uncoating to release biologically active nucleic acid
2
3
45
1
6
Barriers to Successful Gene Therapy
6
Why Gene Therapy Has Not Worked
VIRAL VECTORSmodify a human pathogenic virus to express desired genetoxicunable to be given more than once (innate immunity)less effective in human tissues than initially hoped
NON-VIRAL VECTORS (before Copernicus)toxic in some systemsgenerally less effective than viral vectors
Unimolecular DNA Compaction
Condensation of a Single Molecule of DNA to Form DNA Nanoparticles
Nuclease resistant; stabile in serum Rapid cellular and nuclear uptakeNon-degradative intracellular trafficking pathwayEntry into nucleus of non-dividing cellsHigh in vivo transfection efficiency
DNA Nanoparticle:Component Formulation
DNA
CK30 PEG maleimide
CK30PEG10k
Copernicus Formulation ofPEG-Substituted DNA Nanoparticles
100 nm
PEG-CK30 and plasmid DNAlysine counterion determines shape single DNA moleculereproducible formulationhomogeneous populationno aggregation in saline[DNA] > 12 mg/mlnuclease resistant stable > 3 yearsconsistent in vivo resultstransfect post-mitotic cellsPHASE I TRIAL CF100 nm
TFA Acetate
Stoichiometry of DNA Nanoparticles
8234 bp CFTR expression plasmid + CK30PEG10k
DNA
CK30PEG10k
MOLES PER COMPLEX1
549
16,468 negative charges
MOLECULAR WEIGHT ≅ 13.1 x 106 gm/mol0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
0.0
0.5
1.0
1.52.9 kbp
5.1 kbp
28 kbp
0.0
0.5
1.0
1.5
Input Charge Ratio
Bou
nd C
harg
e R
atio
J. Biol. Chem. 278:32578-32586, 2003
Calculated and Measured Volumes of Compacted DNA Nanoparticles
0 10000 20000 30000 40000 500000
10
20
30
40
502.9 kbp
A1H1
Volume (nm3)
Freq
uenc
y A2 H2
J. Biol. Chem. 278:32578-32586, 2003
Characterization Flow Chart of DNA Nanoparticles
EMA260/A280
TANGENTIALFLOW FILTRATION
EMA260/A280GEL ANALYSISTURBIDITY ANALYSISSALT STABILITYSERUM STABILITYOSMOLALITY, pHENDOTOXINBIOBURDENSTERILITY
CONCENTRATIONDIAFILTRATION
removal ofexcess polycation
only detectable componentis compacted DNA
Nanoparticle Shape is Determined by PolycationCounterion at the Time of DNA Mixing
Trifluoroacetate (TFA) ellipsoidsAcetate rodsBicarbonate rods, toroidsChloride rods (partial)Bromide ellipsoids
IMPORTANT FUNCTIONAL CORRELATESFOLLOWING IN VIVO GENE TRANSFER
In Vivo Gene TransferNanoparticle Optimization
Intrapulmonary
IntradermalIntramuscularIntravenousTopicalIntracranialIntraocular
Polycation compositionPolycation counterionLength of polycation+/- PEGPEG SizePercent PEG substitution+/- Targeting ligands
****
** robust transfection by “non-targeted” complexes
*
*****
Detection of lacZ After Lung Gene Transfer of DNA Nanoparticles
Nova Red stain
5/8mice
3/8mice
No primary AB
Mol. Ther. 8:936-947, 2003
Compacted DNA Nanoparticles:Efficient Transfection of Post-Mitotic Airway Cells
A B
Intranasal
Intrapulmonary
Microinjection of Naked DNA or Compacted DNA Nanoparticles
EGFP expression plasmid Rh-Dextran 155 kD live cells imaged(no fixation)samples blinded beforeanalysis
0.1 1.0 10.0 100.0 1000.00
10
20Compacted Cytoplasm
Compacted Nucleus
Naked Cytoplasm
Naked Nucleus
25
50
75
100
***
******
*
[DNA] (μg/ml)
Gre
en c
ells
%
5 kbp plasmid
J. Biol. Chem. 278:32578-32586, 2003
Microinjection of DNA Nanoparticles of Different Sizes
GFP Plasmids2.9 kbp5.1 kbp (lambda DNA stuffer fragment)10.7 kbp (lambda DNA stuffer fragment)28 kbp (Marek’s virus DNA stuffer fragment)
Equivalent serum stabilities
Major length (nm)
20 30 40 50 60 70 80 9010 100M
inor
leng
th (n
m)
20
30
40
50
10
28
10.7
2.9
5.1
J. Biol. Chem. 278:32578-32586, 2003
15 20 25 30 35 40 45 500
5
10
15 Cytoplasm, Naked DNA
Nucleus, Naked DNAl
Nucleus, Compacted DNA
30
40
50
60
Cytoplasm, Compacted DNA
Minor length (nm)
EGFP
Pos
itive
Cel
ls (%
)
**
Size of Compacted DNA Nanoparticle Determines Nuclear Entry Following Microinjection
J. Biol. Chem. 278:32578-32586, 2003
Effect of DNA Nanoparticle Size on Intrapulmonary Gene Delivery
Ellipsoids
RodsLuciferase Plasmids (kbp)
5.3 9.720.2
λ DNAstuffer
fragments
Plasmid Size and Shape:Intrapulmonary Gene Transfer
20 30 40 50 60 70 80 90
10
20
30
40
50
10 10020 30 40 50 60 70 80 90
10
20
30
40
50
Major Diameter (nm)
Min
or D
iam
eter
(nm
)
TFA Ellipsoids
Ac 5
.3 lu
c
Ac 9
.7 lu
c
Ac 2
0.2
luc
TFA
5.3
luc
TFA
9.7
luc
TFA
20.2
luc101
102
103
104
105
106
log
(RLU
/mg
prot
ein/
pmol
DN
Aab
ove
back
grou
nd)
DNA Nanoparticles
What is the mechanism for cell entry?
DNA Nuclei Merged Image
15 min
30 min
60 min
In Vitro Transfection of Primary HumanTracheal Epithelial Cells
Collaborative studies with Drs. Diane Kube and Pam Davis, CWRU
Uptake and Trafficking ofNon-Targeted DNA Nanoparticles
NON-DEGRADATIVETRAFFICKING PATHWAY little colocalization with antibodies to Rab 5, EEA-1, cathepsin D, or LAMP-1
DNA nanoparticle
nucleolus
In collaboration with D. Kube and P. Davis, CWRUnucleolin
BINDING TO CELLSURFACE NUCLEOLINCOMPLEX
nuclearpore
FITC -- nucleolinRh -- DNA
PRIMARY AIRWAY CELLS
nucleolin
+ -
98% 0%
cell surface
post-translationalmodificationDNA+
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
Clinical Trial -- Cystic FibrosisThe 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
Carriers
Mortality
Treatment
Monogenetic
Source: Cystic Fibrosis Foundation
Prevalence
Correction of Chloride Channel Defect in CFTR -/- Mice
Mouse 3 Mouse 1Pre-treatment Pre-treatment3 Days post rx 3 Days post rx
Mouse 3 Mouse 1
200 sec
Saline treated
12.5mV
CFTR IHC
NPD
collaborative studies with A. Ziady and P. Davis
Copernicus Therapeutics, Inc.
Konstan MW,1 Wagener JS,2 Hilliard KA,1 Kowalczyk TH3, Hyatt SL,3Fink TL,3 Gedeon CR,3 Oette SM3, Payne JM3, Muhammad O3,
Klepcyk P,3 Peischl A,3 Davis PB1, Moen RC3, Cooper MJ3
Single Dose Escalation Study to Evaluate Safety of Nasal Administration of CFTR001 Gene
Transfer Vector to Subjects with Cystic Fibrosis
Department of Pediatrics, Case Western Reserve University School of Medicine, Cleveland, OH 1Department of Pediatrics, University of Colorado Health Sciences Center, Denver, CO 2
3
CF Clinical Trial CTI02001
Phase IPlacebo-controlled, double-blindIN applicationSingle dose, dose escalation3 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)
CFTR Expression Plasmid
pKCPIRCFTRBGH(-)8234 bp
hCFTR
KmR CMV Enhancer
HTLV-Ru5
CMV intron AColE1 origin
BGH polyA
CMV promoter
Standard clinical/laboratory assessmentsNasal washings
baselinedays 2, 3, 4, 7, 14
– cell count– cytokines (IL-6, IL-8)– total protein
Serum IL-6PFTs, CXR, SaO2, CH50, CRP
CF Clinical Trial CTI02001:Toxicity Measurements
No reportable adverse eventsNo adverse events attributed to clinical trial material
(1) grade II pulmonary CF exacerbation at day 14all other adverse events grade I
Mild, transient serum IL-6 rise in 1 subjectNo significant nasal washing IL-6 or IL-8 elevationsNo other laboratory or test abnormalitiesData reviewed by DSMB of CFFTI
CF Clinical Trial CTI02001:Toxicity Measurements
CF Clinical Trial CTI02001:Efficacy Measurements
Nasal potential difference measurementsbaselinedays 2, 3, 4, 7, 14 (or longer if + at day 14)
Nasal cell scrapingsdays 4, 14
– vector DNA (PCR)– vector CFTR mRNA (RT-PCR)– endogenous CFTR and GAPDH (RT-PCR)
Vector DNA Transfection ofNasal Epithelial Cells
SALI
NE
DLI
/II
DN
A D
LI/II
SALI
NE
DLI
II
DN
A D
LIII
0
0.01
0.1
1
10
DN
A C
OPY
NU
MB
ER/C
ELL
Day 14 (both nostrils)
D3 D14
6/6 3/6
6/6 6/6
low
high
DO
SE L
EV
EL
NUMBER OFSUBJECTS + TOTAL
Nasal Potential Difference Analysis
SOP from CFF Therapeutics Development NetworkTracings scrambled and read by impartial observerData finalized before code broken7/126 tracings scored as invalid
catheter movement, excessive signal to noise ratio
Nasal Potential Difference MeasurementsNormal and CF Baseline Values
Standaert, TA et al. Pediatr Pulmonol 37:385-392, 2004
Normal Iso responsemean ± SD = -9.6 ± 5.195% CI = -11.0 to -8.2
CF Iso responseno values < -5 mV
Nasal Potential Difference Correctionm
V
time (min)
Amiloride
Zero Cl ATPIso
Pre-treatment
mV
time (min)
Amiloride
Zero ClATP
Iso
Day 3
-9 mV0 mV0 3 6 9 0 3 6 9
1 min
10 m
V
1 min
10 m
V
Nasal Potential Difference Measurements by Dose Level
DL I DL II DL III0
1
2
3
4≥ -5 mvolts< -5 mvolts
Num
ber
of S
ubje
cts
NPD Corrections
normal95% CI
Data from Both Nostrils
Baseline Days 1-7 Day 13-30
-15
001002003004005006007008009010011012
mean
-10
-5
0
5
delta
NPD
(iso)
(mV)
CF Development Plan
Aerosol development of compacted DNAPromoter refinement of payload plasmidRepetitive dosing studiesSurrogate 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
Battelle Study N104881Cascade Impactor Filters
<0.605
10
15
20
25
30
35 Aeroneb Run # 1Aeroneb Run # 2Pari Run # 1Pari Run # 2
Alveoli ProximalAirw ay
1 2 3 4 5 6 7 8 9 10 11 12 >12Mean Droplet Size (μm)
% o
f Tot
al R
ecov
ered
DNA
DNA Nanoparticle AerosolsRetain Structural and Functional Integrity
before
after
100 nm
100 nm
HepG2 transfection with compacted luciferase DNAbefore or after aerosolization
moc
k
1ng
10ng 1n
g
10ng
100
1000
10000
100000
1000000
10000000
before after
RLU
/mg
prot
ein
PD-IN072503Luciferase Data, Day 2 Collection
saline before after102
103
104
105
106
RLU
/mg
prot
ein
MMAD 4.0 microns +/- 2.1
Plasmid Promoter Evaluation in Mice Following Intrapulmonary Administration
******
*** p<0.001
Collaborative studies with Dr. Deborah Gill and colleagues, University of Oxford
PD-IN102402
2 7 14 28 3510 2
10 3
10 4
10 5RT285 CMVRT277 pUbSaline
Collection Day
Geo
met
ric
Mea
ns o
fR
LU/m
g pr
otei
n (±
SEM
)
Maintenance of Biologic Activity After Repeat Intrapulmonary Dose Administration
PD-IN120503 Repeat Dosing #2Luciferase Data
100
101
102
103
104
105
106
107
RLU
/mg
prot
ein
Group 4
saline x
4Group 3
saline x
3, luc x1 Group 2
saline x
1, CFTR x2
, luc x!
Group 1
CFTR x3, lu
c x1
Dosing Protocol
harvest
Group 1.
Group 2.
Group 3.
CFTR lucCFTR CFTR3 weeks 3 weeks 3 weeks 2 d
CFTR CFTR luc
luc
saline
Group 4.saline
salinesalinesaline
saline
saline
saline
Pipeline Clinical Indications for“Non-Targeted” Nanoparticles
Intrinsic Lung Diseasecystic fibrosisasthma, COPD, α1-AT deficiency, etc.
Lung as Bioreactorhemophilia A and Bcancer (anti-metastases peptides), etc.
Viral Lung Infections (influenza model)Parkinson’s diseaseOphthalmologyCancer Neovasculature Payloads: DNA, RNA, siRNA
Compacted DNA NanoparticlesHighly efficient in vivo gene transfer (post-mitotic cells)
up to 20-fold more efficient than any viral vector in some tissuesEfficient and reproducible formulation manufactured with readily available cGMP raw materials Stable >3 years 4oC; 9 months RTNon-toxic and non-immunogenic; repeat dosing is possibleEncouraging results in human CF clinical trial
NEW CLASS OF THERAPEUTICS WITH ATTRIBUTES OF TRADITIONAL
PHARMACEUTICALS
Counterion of PEG-substituted PolycationInfluences Shape of DNA Nanoparticles
Acetate
TFA
100 nm
100 nm 100 nm
HCO3
100 nm
Chloride
Benign Preclinical Toxicology
High Dose (30-fold higher than highest dose in trial)trace to 1+ mononuclear cell infiltrate around pulmonary veinsno peribronchial or alveolar infiltrates
Low Dose (3-fold higher than highest dose in trial)no histologic findings
no blood test abnormalities, including complementminimal BAL cytokine induction after intrapulmonary dose
no/minimal CpG island response
