Project Two:

Targeting DNA nanoparticles to the Serpin-enzyme complex receptor.

Project References
Assem Ziady, Ph.D., School of Medicine, Case Western Reserve University

The current generation of DNA nanoparticles consists of plasmid DNA condensed with polylysine covalently coupled to polyethylene glycol (PEG-CK30) (1-4). For earlier generation DNA nanoparticles, formed in high salt solution, targeting with a ligand for the serpin-enzyme complex receptor (SEC-R) resulted in 10-1000 fold increased gene expression compared to the non-targeted version depending on system, transgene, time after transfection, and dose (5-10). Similar improvements in stabilized DNA nanoparticles could allow lower doses to give similar results, or limit the cells transfected, avoiding toxicity. Clinical grade DNA is the major manufacturing cost, so reducing the amount needed makes commercial sense. Importantly, once the strategy for addition of targeting ligands is established, other peptides could be substituted and tested, and may present opportunities for introducing DNA into new tissue types (e.g. liver, tumors). The goals of this study are, therefore: 1) to develop DNA nanoparticles targeted at the SEC-R. 2) to test the targeting of these particles in cell culture and in mice. 3) to test whether SEC-R targeting improves duration as well as intensity of expression for stabilized complexes, as it does for first-generation complexes. 4) to test the immunogenicity of the targeted particles.

In this study, we will use SEC-R ligands as prototype for targeting DNA nanoparticles. SEC-R targeting is a good choice because: 1) for the early generation nanoparticles, such targeting both enhanced and prolonged gene expression (7,9), and this sequence, incorporated into the surface of adeno-associated virus, increased reporter gene expression from airway epithelial cells (11). Thus, this sequence is a proven enhancer of uptake and expression in several systems (5-11). Others show that targeting of some abundant receptors enhances gene expression, whereas targeting others does not, even when binding and uptake can be demonstrated (14). SEC-R definitely enhances, in work from three labs (5-11). 2) Our recent studies using fluorescent SEC-R ligands in live cells indicate that they enter the cells rapidly and access the nucleus within five minutes (12,13) 3) Our prior studies show that C105Y is minimally immunogenic by the IV route, and no antibodies were seen in mice dosed via the airway (9).

Initial attempts to add the SEC-R targeting ligand C105Y (CSIPPEVKFNKPFVYLI, binding sequence bold) by 4-PDS chemistry to bifunctional PEG-substituted DNA nanoparticles yielded stable DNA nanoparticles that gave fourfold higher expression than unsubstituted particles administered via the airway. Improvement was less than expected, possibly because C105Y, only 17 aa, might not be long enough to present the binding sequence outside the PEG “shell”. A larger ligand, scFv (Mr ~30 kDa), directed against the polymeric immunoglobulin receptor, attached to PEG-substituted DNA nanoparticles, recognizes its target receptor. To create a longer “arm” for C105Y we will synthesize a polyproline extender of at least the calculated depth of the PEG shell (23nm). Polyproline is selected because poly-L-amino acids are minimally immunogenic, polyproline will create an arm of predictable length (~2nm per residue) (15), so 11 such residues should place the binding sequence outside the “shell”. Alternative “arms” can be made if necessary. Complexes will be tested for binding to HuH7 cells at 4°C, for gene transfer into those cells, and in mice via the airway or IV. Luciferase activities in mice receiving C105Y targeted and unsubstituted nanoparticles will be compared at day 2 post gene transfer. To follow duration of expression for various routes of administration, human Factor IX will be used as reporter gene and blood sampled repeatedly. To identify cell types transfected, mice will be dosed with targeted and unsubstituted nanoparticles encoding lacZ, and tissues evaluated by immunohistochemistry or the X-gal reagent. Immunogenicity will be tested by measuring serum and mucosal antibody response (9), and by testing whether repeat administrations of the gene transfer complex are as effective as the initial dose.

If these experiments are successful, we can define the tissues targeted by the C105Y directed complexes, and plan appropriate indications for their use. For example, if, as we suspect, this complex will target macrophages when given intravenously, then storage diseases such as Gaucher disease might be addressed. Via the airway route, these complexes may prove significantly more efficient than nontargeted complexes and/or direct complexes to different cell types (e.g. alveolar macrophages), and thereby enhance therapy for CF, alpha1-antitrypsin deficiency, factor IX deficiency, or other indications.

Small molecules as payloads

Most drugs now on the market are small molecules. However, many potential beneficial small molecules have not achieved their therapeutic potential because they are too insoluble, too toxic, or need to be concentrated at their primary site of action in order to be effective. Data from Tufts U. indicate that more than half the failures of new drugs are due to bioavailability or solubility issues. Nanotechnology can address these problems. In addition, nanoparticles afford the opportunity to couple localizing tags with therapeutic moieties, so that the location of the therapeutic can be easily verified. The group working in this area consists of Drs. Oleinick, Burda, Kenney, Gao, Boothman, Marchant, and Haaga at Case, Drs. Boiarski, Martin and Grove at iMEDD, Dr. Stout at Ricerca, and Dr. Lentz at Ferro Corp.

We assembled investigators interested in delivery of therapeutic agents, mostly to cancer, but also to inflammatory sites, with several payloads. Dr. Nancy Oleinick, the Joseph T. Wearn University Professor, leads a P01 grant in photodynamic therapy for cancers, and, in ongoing work, has been identifying molecular targets for photodynamic therapy that initiate apoptosis. Two patents covering the initial discovery of the photosensitizer Pc4 and related phthalocyanines have issued, and this compound is licensed for use in sterilization of blood components. Pc 4 is in Phase I trial for dermal malignancies in the UHC Ireland Cancer Center, supported by Oleinick’s P01 and the Cancer Center’s U01: of 3 patients treated at the lowest dose, there were no adverse events, and one patient had a partial response at two different sites. A Phase I trial for cytotoxic T cell cutaneous lymphoma has just been approved, and is the subject of an R21 application. Dr. Oleinick has a longstanding collaboration with Dr. Malcolm Kenney (Dept Chemistry), and recently Dr. Clemens Burda’s expertise has been joined to the group, with the goal of designing improved probes using fluorescent nanoparticles that can target specific sites in or on tumor cells and signal events in their vicinity by energy transfer or other optical techniques. Dr. Kenney holds more than two dozen patents, including one for phthalocyanine photobleach developed with scientists at Procter and Gamble, which is used in household detergents in sunny climes where line drying of clothes is used; it is successful in the hands of consumers. Thus, these investigators have considerable experience not only with photochemistry, but also with its commercialization.

Ricerca, under the leadership of Dr. Stout and Gao wishes to incorporate approved drugs with otherwise limiting solubility or toxicity, such as amphotericin B, into nanoparticles for targeted delivery to appropriate sites, while sparing sites of greatest toxicity. Use of an approved drug facilitates the FDA approval process, and assures that the payload, if properly delivered, will be efficacious. This is, therefore, a sound commercial approach.

One inorganic payload for therapeutic purposes consists of metals such as nickel, coated with platinum, which can be incorporated into nanoparticles and targeted to tumors via addition of antibodies on the surface, or by external magnetic fields. Particles are then activated by inductive heating to kill tumor specifically. Radiologist and inventor John Haaga, M.D., working with C.C. Liu, Ph.D., an engineer expert in biomaterials, has pharmacologic strategies for increasing blood flow to tumors, and others in this nanoparticle group have candidate ligands to anchor these “thermobots” at the target site for activation.

Roger Marchant, Ph.D., Professor of Biomedical Engineering and Director of the Center for Biomaterials, is interesting in limiting restenosis and thrombosis in blood vessels, both native and those that have been instrumented. To this end, with support from NIH, he develops liposomes that are targeted to sites of vascular disruption, have long circulation time, and contain drugs to limit vascular proliferation or thrombosis. These particles capitalize on the differential properties of injured and normal endothelium, and address a major medical problem in the US today.

Ferro Corp (Cleveland OH) has developed proprietary processes for preparation of nanoparticles, many of them via supercritical fluid extractions of emulsions or spray freeze-drying with CO2. This is extremely valuable for scale-up and for solvent-free preparations, and we expect will become important for several projects as they move to animal and clinical trials. Even preparation of the BRTT application has catalyzed interactions among the members of this group and it is anticipated that more will develop as the program unfolds. Three new projects are proposed.

Currently funded projects:

Boothman, D. A. NIH/NCI R01 CA-92250 12/01/01-11/30/05
“Exploiting NQO1 for improved therapy of human breast cancers”.

Boothman, D. A.; NIH/NCI R01 1 R01 CA102792 07/01/03-06/31/08
“Use of ß-lapachone for non-small cell lung cancer therapy”.

Boothman, D. A.; DOD Idea Award PC030740 -01 (# pending) 12/01/03-11/30/06
“Use of ß-lapachone-encapsulated millirods for improved therapy of prostate cancer.”

Burda C, PI. NGT3-52383 Presidential Research Initiative Ohio Board of Regents 02/01/2004-1/31/2005
“Energy Transport in Nanocomposites for Thermoelectric Applications”

Burda C, PI. NSF CHE-0239688 7/1/03-8/31/08
“Study and Control of the Optoelectronic Properties of Ternary Semiconductor Nanomaterials”

Burda C, PI 39881-G5M 7/1/03-8/31/04
"Petroleum Research Fund Main Group Element n-Doped TiO2 Nanostructures for Visible Light Photocatalysis"

J Gao, PI NIH R01 CA90696 (plus minority supplement) 05/01/02-04/30/06
"Interstitial Drug Delivery to Thermoblated Liver Tumors"

J Gao, PI George W. Codrington Foundation 01/01/02-12/31/04
"Polymer Millirods for Intratumoral Drug Delivery to RF Ablated Tumors"

Marchant, RE, PI RO1EB002067-14 (was HL-40047), 4/99 to 3/05 NIBIB
“New Biomedical Interface Materials”

Marchant, RE, PI. NIH,R01 HL-70263,04/02-3/06
“Targeted Liposomal Drug Delivery in Restenosis”

Marchant, RE, co PI NIH, R21 EB-01466 (formerly R21 HL-70263)
Role: Co-PI 07/01/03 - 06/30/05

IBIB
“Self-Assembled Bis-PNA Monomers As New Biomaterials”

Martin, F., PI. NCI Contract number: N01-CO-17043 7/16/01-12/04
“Microfabricated Natural Killer Cells for the Detection and Treatment of Metastatic Tumors.”

Oleinick, N, PI. R01CA83917 6/1/00 – 8/31/07
"Phototherapy of Prostate Cancer –Pro and Anti Apoptosis"

Oleinick, N, P.I. P01 CA48735-7/15/99 – 4/30/05
"Phthalocyanine Photodynamic Therapy: Mechanistic Studies (project and core)"

Oleinick, N, P.I. P20 CA91710-03 (P.I. J. Willson) 9/1/01 - 4/30/05
"In vivo Cellular and Molecular Imaging of Cancer: Imaging of PDT Mechanism by PET"