Project One:

DNA Nanoparticle Therapeutic for Hemophilia

Project References
Mark J. Cooper, M.D., Copernicus Therapeutics, Inc.

Hemophilias A and B are the most common bleeding disorders in males, arising from mutations in X-linked genes encoding for factors VIII and IX, respectively.  There are ~20,000 patients with hemophilia A and B in the US , and ~106,000 patients worldwide (World Federation of Hemophilia website): 80% of these have hemophilia A.  Currently, standard of care is an i.v. infusion of either human plasma-derived or recombinant factor VIII or IX following an acute bleed; the half-life of these proteins in plasma is short (~11 and 19 hrs, respectively), so therapy is limited to the post-hemorrhage setting.  Prophylactic protein therapy to prevent bleeding episodes is not routinely given.  Strategies for prophylaxis would address a current void.  The market for VIII and IX replacement therapy is >$2B per year in the US and Europe.

The Copernicus formulation of DNA nanoparticles efficiently expresses payload genes in lung epithelial cells in mice and humans without induction of limiting toxicities (1-3).  These nanoparticles consist of single molecules of plasmid DNA compacted with 30-mer lysine peptides conjugated to polyethylene glycol.  The small size of these particles facilitates efficient gene transfer (1,4), and transfection efficiencies in murine lung of over 50% are consistently observed.  Importantly, the DNA nanoparticle formulation process is highly reproducible and the nanoparticles are stable in saline at 4oC for over 3 years, at RT for over 9 months, and in serum for hours at 37oC, and give highly consistent results when dosed in the lungs of mice.  When applied to the nasal epithelium of CF knockout mice, statistically significant CFTR chloride channel correction was observed. Several mice had normal CFTR function (and gene transfer efficiency of ~80%).  In a recent phase I clinical trial in cystic fibrosis subjects, compacted DNA encoding CFTR cDNA was applied to the nasal mucosa.  No adverse events were attributed to compacted DNA and 8 of 12 subjects had functional evidence of partial to complete correction of the CFTR chloride channel defect.  Aerosols of compacted DNA having mist sizes optimal for proximal or distal (alveolar) lung delivery have been formulated using several devices, and the post-aerosol compacted DNA retains full structural stability and biological function.  A phase I dose escalation study of compacted DNA aerosols in the lungs of CF subjects is planned for Q205.

The high level gene transfer efficiency of compacted DNA in pulmonary epithelial cells raises the possibility that secreted proteins encoded by these nanoparticles may reach the bloodstream after initial production by the lung.  We further speculate that this process may be optimized if the compacted DNA aerosol is directed to alveolar pneumocytes, since structural barriers to protein diffusion are minimized at the capillary/air interface.  If made in sufficient quantities to achieve therapeutic blood levels, a number of important human diseases could be treated.  In the case of hemophilia, aerosol delivery of compacted DNAs encoding human factor VIII or IX could provide a needleless prophylactic therapy to minimize spontaneous bleeding episodes.  Such a prophylactic therapy is novel and likely would represent a true paradigm shift in medical care for these patients, and may largely supplant the existing markets for factors VIII and IX (except for break-through bleeds which would require intravenous protein therapy). 

In model studies, Dr. Ziady at CWRU dosed C57BL/6 mice with a first generation formulation of compacted DNA encoding human factor IX (hFIX) (5).  This compacted DNA formulation consists of single molecules of plasmid DNA compacted with long chain polylysine (without PEG) and formulated in ~ 1M NaCl. Although technically a different formulation than the PEG-stabilized nanoparticles, both DNA conjugates consist of single molecules of DNA and essentially equivalent gene transfer results in the lungs of mice have been observed.  Following a single 5 μg IV dose of these first generation DNA nanoparticles (targeted with a serpin enzyme complex receptor (SECR) ligand to facilitate intrapulmonary gene transfer), plasma levels of hFIX of up to nearly 250 ng/ml were observed for over a week post gene transfer, whereas non-targeted complexes and naked DNA alone had low or undetectable levels.  This plasma level, ~5% physiologic correction, would be therapeutic in humans with hemophilia.  Following an intrapulmonary dose of compacted SECR-targeted nanoparticles, plasma hFIX levels were ~100 ng/ml.  Of note, this result was obtained with only 5μg DNA (lung plateau dose of PEG-substituted nanoparticles is ~100 μg), and liquid aspirated in the mouse lung is not directed to alveolar pneumocytes.

Because first generation nanoparticles, which do not incorporate PEG, are difficult to formulate and have undesirable solvent and stability characteristics, we propose to develop a hemophilia B therapeutic based on the PEG-stabilized nanoparticle platform.  In initial studies, C57BL/6 mice will be dosed with aerosols of compacted DNA encoding luciferase to standardize and validate dosing parameters using the In-Tox exposure chamber, including measurements of mist droplet sizes using cascade impactors, determination of reproducibility of dosing, and evaluating luciferase activities in mouse lung extracts.  Subsequently, C57BL/6 mice (chosen because antibodies to human FIX are infrequently observed) will receive aerosol doses of PEG-stabilized DNA nanoparticles encoding hFIX, and plasma will be collected over a 2 wk period and assayed by ELISA for hFIX.  If sufficient hFIX is produced, a hFIX immunoprecipitation/Western blot will be run to ensure full length protein is produced.  The next phase will evaluate function of lung-produced hFIX protein in FIX-deficient hemophilia B mice (Jackson Labs, B6.129P2-F9tm1Dws/J) by monitoring the PTT clotting time.  Function of lung-produced hFIX is of interest, since FIX protein is post-translationally modified by gamma-carboxylation, a step that normally occurs in the liver.  Although gamma-carboxylase is widely distributed, including lung (6-8), it is important to confirm that the lung-produced hFIX is biologically active.  If correction of PTT is observed, we will proceed with aerosol dosing of FIX-deficient dogs in the Dept of Veterinary Medicine at Auburn University (Dr. Clint Lothrop).  If this large animal study is encouraging, a clinical development program in support of a phase I dose-escalation lung aerosol trial in hemophilia B subjects will be developed, including formal GLP toxicology and DNA biodistribution studies.  The species in which toxicology data are required will be negotiated with the FDA, although we anticipate a single species (probably rabbit) will be sufficient based on prior experience with our CF development program. Toxicology studies will be performed at Battelle ( Columbus , OH ), because aerosol dosing studies are not feasible at Ricerca.