Second Conference on Drug Delivery and Translational Research, Brooklyn, New York, May 12–13, 2008

INVITATION

2008 Drug Delivery and Translational Research Conference

Dear Colleague, I am delighted to inform you that the scientific program for the Second Conference on Drug Delivery and Translational Research is complete and will be held on May 12–13, 2008 in Brooklyn, New York.

There are four sessions during two days with four speakers each per session. The main focus of discussion is on Gene Delivery. The conference is jointly sponsored by Polytechnic University and SUNY/Downstate Medical Center.

We are looking forward to hearing exciting presentations and participating in discussions on important developments within this research area. To join this event and register, visit www.poly.edu/cddr/registration.

Best regards,

Savio L.L. Woo, Ph.D.

Mt. Sinai School of Medicine

A Phase II Trial of ALN-RSV01, an RNAi Therapeutic for Respiratory Syncytial Virus Akshay K. Vaishnaw, Jared Gollob, Rachel Meyers, Rene Alvarez, John DeVincenzo¹

Alnylam Pharmaceuticals, Inc., Cambridge MA, and ¹Department of Pediatrics, Division of Infectious Diseases, University of Tennessee School of Medicine, Knoxville, TN

Confirmation of RNAi as a widespread physiologic phenomenon in mammalian systems has raised the possibility of pharmacologic intervention using synthetic siRNAs. Before RNAi therapeutics can be evaluated in clinical trials however, they must be designed so that these simple dsRNA species acquire ‘drug-like’ properties. We have made significant progress towards this goal, our most advanced program being ALN-RSV01 which targets the N gene transcript of respiratory syncytial virus (RSV). RSV is a major lower respiratory tract pathogen of young children and accounts for approximately 80% of childhood bronchiolitis cases and 50% of infant pneumonias. Currently, there are no viable treatment options for this infection. The primary site of RSV infection is in the epithelium lining the respiratory tract, suggesting accessibility to an inhaled RNAi therapeutic. In pre-clinical studies ALN-RSV01 substantially inhibited in vitro RSV replication in plaque assays at sub-nM concentrations, and, after direct pulmonary delivery, attenuated lung RSV burden by several log order in both prophylactic and therapeutic mouse models of disease. Rat and monkey toxicology studies have been completed, as have multiple intranasal and inhalational single- and multi-dose Phase 1 human clinical studies. To date, the ALN-RSV01 safety, tolerability and PK profiles look encouraging. The development of anti-virals against respiratory pathogens is further assisted by the availability of experimental infection models. Recently, we have completed a randomized, double-blind, placebo-controlled study evaluating the safety and anti-viral efficacy of ALN-RSV01 in subjects experimentally infected with RSV. The data demonstrate clear evidence of a strong anti-viral effect with good safety and tolerability. In the presentation, I will review the data from prior Phase I safety studies, the current Phase II data and upcoming development plans.

A Phase III Trial of Xrp0038/Nv1fgf for Critical Limb Ischemia Anthony J. Comerota, M.D., FACS, FACC

XRP0038/NV1FGF is a recombinant DNA plasmid encoding for the human acidic FGF-1.¹The plasmid surrounds the FGF-gene and its specific conditional origin of replication backbone, termed Pcor. FGF-1 induces and/or promotes angiogenesis by stimulating cell growth and migration. A Phase I dose escalation trial of 51 patients with critical limb ischemia (CLI) used single and repeated (two) IM doses of NV1FGF.² Patients improved their symptoms; reduced pain (p < .001), reduced ulcer size (p < .01) and improved TCpO₂ (p < .01).

A Phase II study randomizing patients with CLI having non-healing ulcers and no option for revascularization showed that IM NV1FGF 4 mg at two-week intervals x 4 showed similar improvement in ulcer healing as compared to placebo but a significantly longer time to first amputation (p = 0.015) and to first major amputation (p = 0.015).³

The current Phase III study is a randomized, double-blind, placebo-controlled study evaluating four treatments of NV1FGF 4 mg at 2-week intervals in patients with CLI. The primary endpoint is delay in the time to amputation or death in CLI patients with skin lesions who are not suitable for revascularization. Inclusion will be stratified both by diabetes status and by country. Exclusion criteria are similar to those of the Phase II study. Patients will be screened for a 2-6 week period to insure stability of their ischemia and to complete cancer screening, then will enter the trial. Treatment over 6 weeks and follow-up for 12, 18 and 24 months post randomization will be performed.

Ultrasound-Mediated Gene Transfer Zhenping Shen,¹ Andrew A Brayman,² Liping Chen,¹ Baowei Peng,¹ Carol H. Miao^{1, 3}

¹Seattle Children's Hospital Research Institute, ²Center for Industrial & Medical Ultrasound, Applied Physics Laboratory, University of Washington, and ³Dept, of Pediatrics, University of Washington, Seattle, WA

Ultrasound (US) has been shown to be a promising aid to non-viral gene delivery; acoustic cavitation plays an important role in the process. However current methods produce low transfection efficiencies. Stabilized microbubbles (MBs) were utilized to nucleate acoustic cavitation, and lower the acoustic pressure threshold for inertial cavitation occurrence. In order to further enhance the efficiency of US-mediated gene delivery, the use of MBs, ultrasound parameters and plasmid delivery routes were systematically explored to improve gene transfer into the mouse liver. Co-presentation of pDNA,10% MBs, and 1-MHz ultrasound at 4.3 MPa significantly increased luciferase gene expression. Neither US nor microbubbles alone mediated any detectable enhancement of gene expression. Compared with pDNA being delivered by intrahepatic injection to the left liver lobe, intraportal injection delivered pDNA and microbubbles more consistently to the whole liver; with insonation, all lobes expressed the transgene, thus increasing total gene expression. With 4.3 MPa US, gene expression increased with increasing Definity concentration varying from 2.5% to 15% and declined at lower (< 2.5%) or higher (up to 25%) Definity concentrations. Gene expression was also dependent on acoustic pressure over the range of 0 - 4.3 MPa, with a peak effect at 3 MPa. An average of 350-fold enhancement in gene delivery was achieved. The extent of diffuse liver damage was drastically reduced at lower acoustic pressures. The results indicate that coupled with MBs, ultrasound can efficiently and dose-dependently enhance gene expression from pDNA delivered via portal vein injection by an acoustic mechanism of inertial cavitation. Furthermore, US-mediated gene delivery of a liver-specific, high-expressing human factor IX plasmid produced therapeutic levels of factor IX in mice. Therapeutic US in combination with MBs has the potential to promote safe and efficient nonviral gene transfer for the treatment of hemophilia and other genetic diseases.

Intracellular Trafficking of Plasmids, David A. Dean

Department of Pediatrics, University of Rochester, Rochester, NY

The success of viral and non-viral gene delivery relies on the ability of DNA-based vectors to traverse the cytoplasm to reach the nucleus. While the dense latticework of the cytoskeleton impedes free diffusion of large macromolecules, including DNA, transfections do work, and consequently there must be mechanisms by which DNA moves toward the nucleus. Indeed, we and others have shown that plasmids utilize the microtubule network and its associated motor proteins to traffic toward the nucleus. While studying the effects of mechanical strain on the transfection process, we discovered that cyclic stretch causes large scale disruption and reorganization of the microtubule cytoskeleton but at the same time increased gene delivery to the nucleus. Similarly, disruption of microtubules using nocodazole did not abolish cytoplasmic plasmid trafficking. In attempting to understand this apparent paradox, we focused our attention on a pool of stabilized microtubules that are post-translationally acetylated. These microtubules have been shown to play a role in protein traffic within the cell and we asked whether they also play a role in DNA trafficking. Using a variety of approaches, we have found that by increasing the levels of acetylated microtubules we can increase gene transfer, not at the level of transcriptional activation, but rather at the level of cytoplasmic trafficking. Taken together, these results suggest that modulation of the microtubule network can increase the efficiency of gene transfer.

Computer-Assisted Hydrodynamic Delivery of Genes Dexi Liu, PhD

Department of Pharmaceutical Sciences, University of Pittsburgh, School of Pharmacy, Pittsburgh, PA

Hydrodynamic delivery, the application of controlled hydrodynamic pressure in capillaries to facilitate endothelial and parenchyma cell permeability, has become a common method for DNA and RNA delivery in rodents and proven useful for a wide range of applications. For example, hydrodynamic tail vein injection of plasmid DNA has been used to assess the involvement of specific genes in development or regression of pathophysiological conditions. It has also been used for evaluating the activity of regulatory sequences that control gene expression under the physiological condition in a whole animal. In addition, more recent success in demonstrating long-term gene expression with gene product at therapeutic level in small animals has provided great incentive for establishing a hydrodynamic procedure clinically applicable. Toward this end, we have developed a computer-assisted injection device capable of performing hydrodynamic gene delivery in various organs and in small and large animals. Our system consists of a computer, a solution driver, a pressure transducer capable of detecting real time pressure at the injection site of the targeted organ, and a magnetically controlled switch. The injector achieves the precise and real time control over an injection by regulating the on/off switch at the solution driver through a negative feedback circuit looped by the pressure transducer via the computer. Depending on the pressure profiles programmed in the computer, the injection device injects DNA solution into the animal through a catheter when the pressure is below the desirable pressure and shuts off when the desirable pressure is reached. Using the liver, kidney and muscle as the targeting organs in mice, rats and pigs, we demonstrated a full control over injection with our computer-assisted injection device. Continuous injection with or without interval time was also achieved. When assessed by reporter plasmids, we have established the relationship between the pressure profile of each individual injection and the level of reporter gene expression. This presentation will focus on our recent progress toward establishing optimal parameters for liver gene delivery using pigs as an animal model. The perspectives on clinic use of this new injection device for human gene therapy will also be discussed.

Dynamic PolyConjugates: Synthetic delivery systems forin vivo delivery of siRNA Jon A. Wolff,^1,2 David B. Rozema,¹ David L. Lewis,¹ Darren H. Wakefield,¹ So Wong,¹ Jason J. Klein,¹ Paula L. Roesch,¹ Stephanie L. Bertin,¹ Tom W. Reppen,¹ Qili Chu,¹ Andrei V. Blokhin,¹ and James E. Hagstrom¹

¹Mirus Bio Corporation, Madison, WI; ²University of Wisconsin-Madison, Madison, WI

The efficient delivery of a variety of nucleic acids such as plasmid DNA and siRNA to the appropriate target cell in animals has been a challenge. The development of such technology would be important both as a research tool and therapeutic method. The rational design of synthetic delivery systems are based upon the current paradigm for nucleic acid delivery in which the delivery process is broken into discrete transport steps. For siRNA delivery to cells in vivo, the two essential steps are cell targeting and entry into the cytoplasm so as to accesses the RISC complex. Targeting requires not only a ligand that recognizes a receptor on the target cell but also polyethylene glycol (PEG) to prevent non-specific interactions with non-target cells, blood components and other extracellular elements. Cellular entry requires the use endosomolytic agents to disrupt the endosomes. The challenge has been to incorporate these various elements for targeting and endosomal release into a siRNA delivery complex that has in vivo activity. Often the elements that enable targeting, such as PEG groups, inhibit endosomal release. We have recently integrated these components and strategies together to enable efficient and non-toxic delivery of siRNA's to hepatocytes and other cells in mice. A key element of this new approach is the use of masked endosomolytic agents (MEA's) that improve the release of nucleic acids from endosomes. When the MEA enters the acidic environment of the endosome, a pH-labile bond is broken releasing the agent's endosomolytic capability. The use of a MEA within polymeric conjugates of siRNA has enabled the efficient delivery of siRNA into hepatocytes in vivo. Using this delivery system and simple, low pressure tail vein injections, we have demonstrated effective knockdown of two endogenous genes in mouse liver: apolipoprotein B (apoB) and peroxisome proliferator-activated receptor alpha (ppara). Fatty livers (steatosis) were observed in animals receiving siRNA against apoB, a clear phenotypic change. The delivery system was well tolerated with no significant changes in serum liver enzyme or cytokine levels. In summary, the use of labile bonds to mask endosomolytic agents provides a critical design feature for enabling efficient in vivo delivery without sacrificing endosomolytic function. This is a modular platform system so other ligands could be easily incorporated to enable targeting to other cells, an avenue that is under investigation.

Delivery of siRNA with Lipopolyplex Nanoparticles Leaf Huang, PhD

Division of Molecular Pharmaceutics, School of Pharmacy, University of North Carolina, Chapel Hill, NC

Systemic delivery of siRNA remains a challenge due to the high hydrophilicity and low stability of siRNA in vivo. We have developed a novel lipid based nanoparticles that delivered siRNA predominantly (70-80% injected dose/g tissue) to the tumor when intravenously administered. Pharmacokinetics and tissue distribution studies showed the tumor was the major uptake organ for siRNA formulated in the PEGylated nanoparticles. A small molecule targeting ligand (anisamide) attached to the distal end of PEG increased the cellular internalization when nanoparticles extravasated into the tumor tissue. After 3 daily injections (1.2 mg/kg), the siRNA in the targeted, PEGylated nanoparticles almost completely silenced the target gene (EGF receptor) in the entire tumor. Using a pulmonary metastasis model of mouse melanoma B16F10, we have shown that the ED50 of the injected siRNA can be as low as 75 μ g/kg, demonstrating the potency of the delivery system. Simultaneous delivery of three siRNA against three different target genes has resulted in a significant tumor growth inhibition and prolongation of the survival without any detectable inflammatory toxicity which is commonly seen with the double-stranded RNA.

A Phase 2 Repeat Dosing Clinical Trial of SB-509 in Subjects with Diabetic Neuropathy Marc Rendell, M.D. and Dale Ando M.D.

Creighton Diabetes Center, Omaha, NE, and Sangamo Inc, Oakland, CA

C2H2 zinc fingers are found in 2% of all human genes, and they are by far the most abundant class of DNA-binding domains found in human transcription factors. SB = 509 is a plasmid containing a sequence encoding a zinc finger transcription factor for VEGF A. We hypothesize that local injection of SB-509 along the course of major sensory and motor nerves will result in an increase in local VEGF levels, leading to microvascular growth in and around nerve bundles. The resultant microvascular support may improve the structure and function of nerves, protecting and repairing the damage caused to nerves by diabetic neuropathy.

Primary Objectives: To compare in subjects with diabetic neuropathy the effect of SB-509 versus placebo on a pre-defined multi-endpoint analysis that includes visual analog scale for pain intensity (VASPI), total neuropathy score (TNS), nerve conduction velocity (NCV), quantitative sensory testing (QST), and epidermal nerve fiber density (ENFD) and regeneration (ENFDR).

Secondary Objective: To evaluate the safety of SB-509 as compared to placebo in subjects with diabetic neuropathy. A double blind placebo controlled multicenter trial of local injection of SB-509 in approximately 100 patients with diabetic neuropathy has begun. The duration of treatment is 5 months with three injections over that period. There will be a followup period of 7 months to assess response. At the present time, the trial is fully enrolled with completion scheduled in second quarter 2009.

Gene Delivery by Poly-lysine Conjugated DNA Nanoparticles Mark J. Cooper, M.D.

Copernicus Therapeutics, Inc

A safe and effective non-viral nanoparticle technology has been developed by Copernicus Therapeutics that compacts single molecules of nucleic acids with polycationic carriers, such as polyethylene glycol (PEG)-substituted lysine 30-mer peptides. These nanoparticles are non-immunogenic, non-inflammatory, and non-toxic, and transfect various non-dividing tissues in vivo with efficiencies comparable to viral vectors. DNA nanoparticles achieve transfection efficiencies of 30% to 80% of surface epithelial cells in the lung, are equivalent in efficiency to viral vectors in the brain, and transfect up to 99% of retinal photoreceptors after local eye delivery. Nanoparticles can be repetitively administered to the lungs of mice without any reduction of transgene activity. The gene transfer efficiency is probably related to the unimolecular nature of the DNA nanoparticle, resulting in an effective diameter of < 12 nm, and to its association with cell surface nucleolin, which facilitates efficient cellular internalization, non-degradative (non-lysosomal) intracellular trafficking, and nuclear uptake. Based on its safety profile and gene transfer activity, Copernicus initiated a program for CFTR replacement therapy to the lungs of patients with cystic fibrosis (CF). In an intranasal single dose escalation phase I/IIa human clinical trial in CF subjects, there were no adverse events attributed to the nanoparticles and 8/12 subjects had partial to complete functional correction of the CFTR chloride channel. In preparation for a lung aerosol trial in CF subjects, DNA nanoparticle aerosols have been shown to be stable and to effectively transfect the lungs of intubated rabbits. Following a single dose to the lungs of mice, hCFTR mRNA expression is > 100% of endogenous mCFTR and expression persists for at least 2 months. In other studies, subretinal delivery of compacted DNA prevented blindness in a mouse model of retinitis pigmentosa. In summary, these findings suggest that compacted DNA nanoparticles may provide a safe and effective delivery system for treating the lung manifestations of cystic fibrosis and have potential for treating various disorders of the lung, eye, and brain.

Electroporation-Based Cancer Gene Therapy Richard Heller

Department of Molecular Medicine and Center for Molecular Delivery, University of South Florida, College of Medicine, Tampa, FL

A critical aspect of gene transfer is effective delivery of the transgene to the appropriate target. Both biological (viral) and non-viral approaches have been evaluated. A drawback of non-viral is ineffective expression of the transferred gene. This is typically related to the inefficient delivery of the plasmid DNA. Electrically mediated delivery (electroporation) of plasmid DNA is quickly being accepted as a viable approach to achieve effective delivery. The versatility inherent in this delivery system is an important reason for this growth. Careful selection of tissue target and delivery parameters including electrode and electrical conditions allows an investigator to obtain the type of transgene expression necessary for a particular therapeutic application. Many investigators are using this delivery approach to deliver agents with anti-tumor potential. Most of the pre-clinical therapeutic studies of direct in vivo EP demonstrate significant tumor regression, indicating that the delivered genes are potentially effective as anti-tumor agents. A limited number of studies have demonstrated long-term, complete tumor regression, including studies involving delivery of plasmids encoding IL-12 as a single agent in melanoma and SCC or IFNα as a single agent in SCC or melanoma. The true evaluation of this delivery approach as a means for moving from gene transfer to gene therapy will be based on the results from clinical studies. The first clinical study was initiated in late 2004 at the H. Lee Moffitt Cancer Center and Research Institute (Tampa, FL). This was a phase I dose escalation trial in patients with accessible subcutaneous metastases with melanoma. Following the initiation of this trial several other clinical trials utilizing electroporation to deliver plasmid DNA have been initiated. Once results from these studies are released, electrically mediated plasmid delivery will either expand with additional trials or it will regress to use as a preclinical or laboratory tool to evaluate potential targets.

An Apo A-I-Conjugated Nanoparticle System for Hepatic Gene Delivery Meehyein Kim

Mogam Research Institute, South Korea

Currently, there are two major hurdles to achieving clinically usable small interfering RNA (siRNA)-based therapeutics: establishment of an efficient delivery system that transfers the siRNA to the correct tissue(s); and the reduction of unintended immunotoxicity associated with unmodified siRNA. In this presentation, the potential of apolipoprotein A-I (apo A-I) for the systemic delivery of cationic liposome vehicles containing siRNA to the liver is demonstrated. As a proof-of concept, synthetic siRNA targeting hepatitis B virus (HBV) were formulated with apo A-I-bound DOTAP/cholesterol liposomes (DTC-Apo), and injected intravenously into mice carrying replicative HBV. The results show that administration of these nanoparticles significantly reduces viral protein expression by about 70% only at a single dose (≤2 mg siRNA/kg). The major advantage of this system is that apo A-I as a liver-targeting moiety is a natural serum protein and functions as a benefit factor to decrease cholesterol levels, and thus does not induce immunological or histochemical toxicity. The liver-targeted gene silencing was confirmed by in vivo images in which bioluminescent signals emitted from the liver were efficiently reduced after intravenous administration of luciferase-specific siRNA and DTC-Apo lipoplex. Moreover, it was detected that systemic injection of an unmodified HBV siRNA encapsulated within DTC-Apo activates the innate immune response in mice. However, 2′-O-methy (2′-OMe) modification of siRNA sense-strand uridine or uridine/adenosine residues efficiently abrogated the immunostimulatory properties, and still maintained the gene silencing potency. Interestingly, pyrimidine modification by 2′-OMe or 2′-fluoro (2′-F) substitution failed to circumvent liposome-induced immune recognition. Thus, it is expected that our unique approach to siRNA delivery not only creates a foundation for the development of a new class of RNAi-based therapeutics against hepatitis viruses or hepatic genes related to tumor growth, but also provides useful information for the design of chemically-modified siRNAs for in vivo applications.