Robert S. Langer - US grants

DESCRIPTION (provided by applicant): This is a grant renewal for studies on formulations of high molecular weight agents, such as proteins and nucleic acids. For the past 29 years, this particular grant has been central for our drug delivery research. We believe the impact of our NIH funded research in this area has been significant and that our work has helped provide some of the fundamental concepts necessary for the clinical development of controlled release of macromolecules and for localized drug delivery. Examples of such delivery systems are Lupron Depot, Gliadel and Nutropin Depot. In writing our last competing renewal grant proposal we felt that much of the fundamentals of peptide and protein release had been established and that the greatest impact could come from extending our research nucleic acid delivery. Our studies in this area have led to a greater mechanistic understanding, and have resulted in 35 peer-reviewed papers, including in such journals such as Science and Nature. The major barrier to the success of gene therapy in the clinic is the lack of safe and efficient DNA delivery methods. Modified viruses, while effective at transferring DNA to cells, suffer from serious toxicity and production problems. In contrast, non-viral systems offer a number of significant potential advantages, including ease of production, stability, low toxicity, and reduced vector size limitations. From our studies we have discovered that optimal intracellular delivery to different tissues in the body requires different materials. Therefore, we propose to develop combinatorial libraries of new materials with the specific goal of generating clinically useful, non-viral methods for gene therapy. Lung cancer will be used as a model disease. We propose to explore two complementary approaches: 1) developing lung-tumor cell specific DNA delivery systems for the anti-tumor therapy and 2) developing anti-lung cancer DNA vaccines by targeting and activating native immune cells. Our specific aims are: 1. To develop next-generation biodegradable polymers for use as efficient and non-toxic vectors for lung cancer and DNA vaccines. 2. Development of cell specific DNA delivery systems for lung tumor and dendritic cell targeting. 3. To test the in vivo gene delivery efficiency of biodegradable vectors composed of PEI- and PBAE- based polymer and that of their electrostatic ligand coated ternary polyplexes in normal and lung cancer mouse models. 4. To examine potential of PEI- and PBAE- based vectors for their use as DNA vaccines.

The controlled release of marcomolecules from inert polymeric martix devices has been extensively researched by our group. Furthermore, a recently synthesized class of bioerodible polymers, made from the non-peptide linking of typrosine dipeptide derivatives, demonstrates adjuvanticity in both monomeric and polymeric form. The aim of the proposed research is to develop a controlled release immunization implant which will have a two-fold effect. First, the device will produce sustained, controlled release of antigen in vivo, thereby initiating production of antibody specific for that antigen and successfully immunizing the animal. Second, the products formed during polymer hydrolysis will have an adjuvant effect on the immune response, thus enhancing the level and duration of antibody production and improving protection against subsequent challenge. To achieve this goal: 1) Poly(iminocarbonates) composed of N- and C-blocked derivatives of N-(L-tyrosyl)-L-tyrosine will be synthesized and characterized with regard to molecular weight mechanical properties, surface properties, and degradation behavior; 2) The release of model antigens from devices composed of these polymers will be measured and mathematically modelled; 3) Adsorption-desorption studies of polymer and antigen will be conducted to investigate the "depot" effect mechanism of adjuvanticity; 4) Polymeric devices containing antigen and polymer-antigen adsorbates will be implanted or injected in mice, and the antigen- specific antibody levels will be measured by ELISA; and 5) Release patterns from devices will be altered with ultrasound or formulation parameters to determine the effect of dosage on adjuvanticity.

This is a competing renewal proposal of our previous grant application. The objective of the previous grant application was to investigate the effects of the use of ultrasound to enhance polymeric drug delivery and drug permeation through skin. In this past grant period, we investigated the effects of polymer molecular weight, cavitation, temperature, solution gas content, and other factors on ultrasound induced polymeric drug delivery; we showed that ultrasound could enhance transdermal drug delivery, that it could act as a trigger to terminate the activity of controlled release implants, and that it could provide a new rapid method of achieving protein blotting from electrophoretic gels. We believe that these studies will have wide applicability in a number of areas for ultrasound-assisted transport and, indeed, many of these avenues of application are now being pursued in other laboratories. In the current grant application, we wish to focus our attention on one of these areas- ultrasound-assisted transdermal drug delivery with special emphasis on protein delivery in part due to the reviewers' previous comments-because of the great impact that can now be made in this area. The specific goals of this proposal include: 1. Understanding the mechanisms of transdermal flux enhancement due to ultrasound. 2. Understand the dependence of sonophoresis on ultrasound parameters. 3. Investigate safety issues of sonophoresis. 4. Modeling ultrasound-mediated enhancement and transdermal fluxes.

This is a competing renewal proposal in the area of the development of controlled release systems for immunocontraception. There are several reasons why this is a very important area. First, for active immunization with sperm antigens, the ability to release the antigens for long periods of time will enhance it's immunogenicity. Second, for passive immunization with monoclonal antibodies against sperm or zona pellucida protein, the ability to provide long-term release of an active antibody would be very desirable. In the past grant period, we proposed the synthesis of a class of novel degradable polymers involving L-tyrosine peptide derivatives. The major objective was to design devices that would provide controlled release of the antigen for prolonged periods of time. In addition, one of the desirable features of these polymers would be that the products formed from polymer hydrolysis could have an adjuvant effect on the immune response and thereby enhance the level or duration of antibody production. In the last grant period, we successfully synthesized polymers composed of, or containing, a variety of tyrosine and tyrosine dipeptide monomers and fully characterized them with respect to a variety of properties relevant to antigen delivery. We developed micron-sized antigen-loaded spheres (i.e., microspheres) from these polymers and developed approaches to provide prolonged release of model proteins from these systems. A number of papers in very good journals have resulted from these studies. The specific aims of the current proposal are to further develop this area by utilizing specific antigens (such as PH-20, a peptide from the active site of a fertilin beta, and a recombinant fertilin beta extracellular domain) now available from our collaborators, and to expand potential methods of achieving controlled release immunocontraception, including new routes of administration and approaches for achieving pulsatile release. The specific aims are as follows: 1) To incorporate antigens from the other Centers into polymer microspheres based on tyrosine derivatives. 2) To pursue the idea of vaccine delivery by using a new approach involving FDA approved lactic/glycolic acid polymers which can provide pulses of vaccine. In addition, we will pursue a novel idea of stabilizing proteins in these polymers systems using an oil-based microencapsulation. 3) To develop delivery systems for mucosal immunization, in particular systems which can be administered by the pulmonary route.

DESCRIPTION: (Adapted from the applicant's abstract) The concept and practice of seeding cells onto degradable polymer scaffolds, such as poly(lactic acid) (PLA) or poly (glycolic acid) (PGA), in vitro followed by eventual in vivo implantation has become an important methodology in tissue engineering. These scaffolds provide structural support for the cells until they develop their own extracellular matrix, but scaffold mechanical properties have yet to be specifically designed to provide an environment that will match that of the native tissue. The specific interest of this project is the engineering of cardiovascular tissues. In the investigators' experiences with existing PLA or PGA tubular and valvular leaflet scaffolds, it has been difficult to sew them into the native tissue, bend them to fit a desired or required conformation within the animal, or replace all three pulmonary valve leaflets without leading to unacceptable valvular stenosis. The investigators' goals are to develop the next generation of tissue engineering polymer scaffolds with mechanical properties (e.g. elasticity) that closely model those of the native tissue (pulmonary valve leaflets, arteries) and to investigate the influence of such an environment on cell proliferation, tissue formation, and function of engineered tissues in vivo. Their hypothesis is that significant advances in tissue engineering may be realized if the biomaterials to be used as scaffolds can be specifically designed to resemble the mechanical properties of the native tissue in question. Accordingly, the Specific Aims are: 1) Synthesis and characterization of elastic, degradable polymers with appropriate mechanical properties for use as cardiovascular tissue engineering scaffolds; 2) Test the degradation profiles of the new polymers and monitor the changes in mechanical properties over time; 3) Test the polymer-cell compatibilities and interactions (attachment and spreading) on polymer films using appropriate cell types (e.g. endothelial cells, smooth muscle cells, fibroblasts). Cells will be provided by Dr. Mayer at Children's Hospital; 4) Select best candidates based on Aims 1-3, then design and prepare scaffolds in the shapes of tubes for blood vessels and leaflets or whole valves for heart valves; 5) Carry out in vitro cell seeding studies to determine the influence of polymer properties and bioreactor conditions on cell differentiation, proliferation, and tissue formation in conjunction with Drs. Vacanti, Bischoff, and Roth at Children's Hospital; 6) Implantation of cell-scaffold devices at the appropriate time frame based on the findings of Aim 5 in small animal models (to be carried out by Drs. Mayer and Vacanti at Children's Hospital).

It is well known that the method by which a drug is delivered can have a significant effect on the drug's therapeutic efficacy. Most drugs have a concentration range in which they have maximum efficacy. Conventional drug delivery regimens result in sharp changes in systemic drug levels that can be fatal. Controlled drug delivery can alleviate the problems associated with conventional therapy by providing stable drug bioavailability in a therapeutically meaningful range and in addition can be used to localize the therapy to the tissue site of interest. We recently showed that it is possible to fabricate a solid-state silicon microchip in which a number of chemicals or drugs can be stored and released on demand by an external trigger. One advantage of this novel controlled release system is that it allows for simultaneous release of multiple drugs in complex release profiles. One can potentially develop a device that can be pre- programmed to delivery combination drugs in a pre-determined fashion. We believe that this novel delivery technology has broad utility in the biomedical area such as local delivery of anesthetics for pain management, sub-dermal delivery of vaccines, periodontal delivery of antibiotic and anti-inflammatory agents, localized delivery of anti-tumor and neoplastic agents, gene delivery, delivery of antiarrhythmic agents to name a few. Based on the above mentioned rationale and our preliminary results we propose the following specific aims: (1) Development of an active, silicon based microchip for controlled release of drugs that can operate autonomously, (2) Development of a passive, polymeric chip for the controlled release of drugs, (3) Evaluate the biocompatibility of active and passive microchip delivery device and (4) Evaluate the drug release both in vitro and in vivo, specifically: (a) show that predictable drug release is possible from both active and passive microchips (b) study a pathology such as brain tumors that maybe be better treated by combination therapy (c) evaluate the efficacy of these devices in this tumor model.

DESCRIPTION (provided by applicant): We propose to use our established and well documented history in the development of biomaterials as scaffolding for tissue replacement as a platform for stem cell approaches in craniofacial tissue regeneration. The governing hypotheses of this work include: i) that human stem cells of embryonic origin can be differentiated into craniofacial tissue producing cells such as osteoblasts and chondrocytes, ii) that highthroughput techniques can be used for the screening of large numbers of media and polymer candidates for potential use with tissue engineering scaffolds, and iii) that the appropriate combination of stem cells with 3-dimensional polymer scaffolds can be used for the production of complex craniofacial tissues. To test these hypotheses, we propose the following: Aim 1: Examine the in vitro differentiation of human embryonic stem cells into precursors of craniofacial tissues. Our previous work investigating the in vitro differentiation of human embryonic stem cells into various cell types and tissues will be continued to assess conditions necessary for differentiation into cell types necessary for craniofacial tissue formation. Aim 2: Investigate the interaction of human stem cells with polymer surfaces using high-throughput screening technology. Recently, we have developed techniques for the rapid screening of cell/polymer interactions using nanoliter-scale microarrays of various monomers and polymers. Success will be assessed through cell proliferation and the presence of tissue specific markers for craniofacial tissues. Aim 3: Assess in vitro tissue production by differentiated and undifferentiated human embryonic stem cell seeded polymeric scaffolds incorporating various genes and growth factors. Polymeric materials that support the differentiation of human stem cells into craniofacial tissues will be fabricated into 3-dimensional scaffolds incorporating various genes and growth factors and analyzed by histological staining of various markers. Aim 4: Optimal candidates from the previous aims will be seeded with differentiated and undifferentiated cells and implanted either subcutaneously or in a critical sized defect model in rats. The subcutaneous model in athymic rats will be used to assess tissue production by stem cell seeded scaffolds in an in vivo environment, including scaffolds with cells differentiated into multiple phenotypes. The cranial defect model in athymic rats is a clinically relevant model that is commonly used as a measure of bone regeneration.

DESCRIPTION (provided by applicant): The overall objective of this proposal is to develop a fundamental understanding of the role of localized transport regions (LTRs) in low-frequency sonophoresis (LFS), with an emphasis on: 1. Understanding if the LTRs are Indeed Regions of Highly-Localized Transdermal Transport for both Hydrophilic and Hydrophobic Permeants: Specifically, demonstrating whether the LTRs are regions of high permeability compared to the surrounding regions of ultrasound-treated skin (the non-LTRs) for a broad class of permeants. 2. Understanding the Mechanisms that Govern the Formation of the LTRs in LFS: Specifically, understand: i) the role of the surfactant sodium lauryl sulfate (SLS) in LTR formation, and ii) the roles of acoustic cavitation, the ultrasound acoustic field, and the skin surface topography in LTR formation. 3. Understanding the Nature of the Permeation Pathways that Exist within the LTRs and the Non-LTRs: Specifically, identify the type of permeation pathway (transcellular, intercellular, or a combination of both) followed by permeant molecules traversing the skin within the LTRs and the non-LTRs. 4. Investigating the Safety of LTR Formation on Skin Treated with LFS: Specifically, determining: i) the reversibility of LTR formation in the skin, and ii) the biological effects of LTR formation. 5. Mathematical Modeling of Transdermal Transport in the Presence of LTRs: Specifically, i) developing mathematical models to more accurately evaluate experimental transdermal permeabilities across ultrasound-treated skin with LTRs, and ii) developing models to confirm the presence of transcellular and/or intercellular transdermal pathways within the LTRs and the non-LTRs in the stratum corneum resulting from the ultrasound treatment.

DESCRIPTION (provided by the applicant): Not provided.

DESCRIPTION (provided by applicant): We propose to use our established and well documented history in the development of biomaterials as scaffolding for tissue replacement as a platform for stem cell approaches in craniofacial tissue regeneration. The governing hypotheses of this work include: i) that human stem cells of embryonic origin can be differentiated into craniofacial tissue producing cells such as osteoblasts and chondrocytes, ii) that highthroughput techniques can be used for the screening of large numbers of media and polymer candidates for potential use with tissue engineering scaffolds, and iii) that the appropriate combination of stem cells with 3-dimensional polymer scaffolds can be used for the production of complex craniofacial tissues. To test these hypotheses, we propose the following: Aim 1: Examine the in vitro differentiation of human embryonic stem cells into precursors of craniofacial tissues. Our previous work investigating the in vitro differentiation of human embryonic stem cells into various cell types and tissues will be continued to assess conditions necessary for differentiation into cell types necessary for craniofacial tissue formation. Aim 2: Investigate the interaction of human stem cells with polymer surfaces using high-throughput screening technology. Recently, we have developed techniques for the rapid screening of cell/polymer interactions using nanoliter-scale microarrays of various monomers and polymers. Success will be assessed through cell proliferation and the presence of tissue specific markers for craniofacial tissues. Aim 3: Assess in vitro tissue production by differentiated and undifferentiated human embryonic stem cell seeded polymeric scaffolds incorporating various genes and growth factors. Polymeric materials that support the differentiation of human stem cells into craniofacial tissues will be fabricated into 3-dimensional scaffolds incorporating various genes and growth factors and analyzed by histological staining of various markers. Aim 4: Optimal candidates from the previous aims will be seeded with differentiated and undifferentiated cells and implanted either subcutaneously or in a critical sized defect model in rats. The subcutaneous model in athymic rats will be used to assess tissue production by stem cell seeded scaffolds in an in vivo environment, including scaffolds with cells differentiated into multiple phenotypes. The cranial defect model in athymic rats is a clinically relevant model that is commonly used as a measure of bone regeneration.

DESCRIPTION (provided by applicant): We propose to use our established and well documented history in the development of biomaterials as scaffolding for tissue replacement as a platform for stem cell approaches in craniofacial tissue regeneration. The governing hypotheses of this work include: i) that human stem cells of embryonic origin can be differentiated into craniofacial tissue producing cells such as osteoblasts and chondrocytes, ii) that highthroughput techniques can be used for the screening of large numbers of media and polymer candidates for potential use with tissue engineering scaffolds, and iii) that the appropriate combination of stem cells with 3-dimensional polymer scaffolds can be used for the production of complex craniofacial tissues. To test these hypotheses, we propose the following: Aim 1: Examine the in vitro differentiation of human embryonic stem cells into precursors of craniofacial tissues. Our previous work investigating the in vitro differentiation of human embryonic stem cells into various cell types and tissues will be continued to assess conditions necessary for differentiation into cell types necessary for craniofacial tissue formation. Aim 2: Investigate the interaction of human stem cells with polymer surfaces using high-throughput screening technology. Recently, we have developed techniques for the rapid screening of cell/polymer interactions using nanoliter-scale microarrays of various monomers and polymers. Success will be assessed through cell proliferation and the presence of tissue specific markers for craniofacial tissues. Aim 3: Assess in vitro tissue production by differentiated and undifferentiated human embryonic stem cell seeded polymeric scaffolds incorporating various genes and growth factors. Polymeric materials that support the differentiation of human stem cells into craniofacial tissues will be fabricated into 3-dimensional scaffolds incorporating various genes and growth factors and analyzed by histological staining of various markers. Aim 4: Optimal candidates from the previous aims will be seeded with differentiated and undifferentiated cells and implanted either subcutaneously or in a critical sized defect model in rats. The subcutaneous model in athymic rats will be used to assess tissue production by stem cell seeded scaffolds in an in vivo environment, including scaffolds with cells differentiated into multiple phenotypes. The cranial defect model in athymic rats is a clinically relevant model that is commonly used as a measure of bone regeneration.

7. TRAINING, EDUCATION, KNOWLEDGE TRANSFER 7.1 Training This CCNE will function to train the next generation of scientists and engineers in the use of the tools of nanotechnology to understand and ameliorate diseases, including cancer. Specifically, through the research projects funded by the Center as well as the associated educational activities, we will train graduate students and postdoctoral fellows at the interface between nanotechnology and cancer biology. We believe strongly that a true integration of different disciplines is best achieved by the joint training and supervision of trainees so that they become expert in all relevant areas of study. Accordingly, we place significant emphasis on efforts to attract students with backgrounds in cancer biology and basic cell and molecular biology as well as those from chemistry, physics andengineering disciplines. The CCNE will offer an ideal environment for the cross-training of such individuals, and we will work with the graduate and other training programs at the represented institutions to ensure that the students and fellows are able to perform their work in this cross-disciplinary setting.. In fact, there is a large pool of excellent students and MIT/Harvard already have in place administrative and educational mechanisms for encouraging interdisciplinary activities among these students and the respective faculty members. The training component will include participation in a CCNE-specific lecture series, classroom training and workshops. At both institutions, there are multiple courses with "nano" in their titles and several more that are closely related to the fields of study in the CCNE. Similar courses are also offered at Harvard. The educational component of the CCNE will provide support for the interdisciplinary training of 2 graduate students and 2 postdoctoral fellows per year and 5 undergraduates. This is in addition to the individuals funded directly by the projects and pilot projects Overall, we estimate to train 15-20 students and an equal number of post-doctoral fellows per year in the CCNE. 7.2 Interface with other nanotechnology training programs Harvard's Nanoscale Science and Engineering center (NSEC;PI: Westervelt) has an extensive educational and outreach program that has been in existence for 5 years. The program (for details see http://www.nsec.harvard.edu/education.htm) has lecture series, webcasts, interactions with the Museum of Science, cablecasts via New England Cable News, fellowships for women and minorities, undergraduate programs, K-12 Teacher programs and knowledge transfer programs to the public. We will harness these existing mechanism and in close collaboration with Dr. Westervelt plan for joint programs in the future open to participants of both programs. A second existing training program is that of MIT's Institute of Soldier Nanotechnologies (ISN;PI: Ned Thomas) and which will be tightly integrated with this consortium. There are currently over 130 graduate students and nearly 40 postdoctoral fellows working on different projects of the ISN. All standard MIT research center communications are implemented: web site, newsletters, brochure, video, hosting of groups for overviews and tours (everyone from school kids to alumni to four-star generals). There is a monthly seminar series, a K-12 outreach program, and contributions to the MIT Museum's family program. 7.3 Interface with NIH training programs There also exist a number of training programs associated with members of the CCNE that focus on cancer biology, biology, biophysics, materials engineering, physical sciences. This includes training grants in the MIT Biology Department (5 T32 GM07287-30), Computational Systems Biology Initiative (1-T90- DK070114-01, 1-R90-DK071503-01), Division of Biological Engineering (5-T32-ES070-2O), Chemistry Department (T32-ES-07020, T32-CA-09112). Also included are a T32 training program at CMIR (T32 CA79443), a P50 associated Career Development program (P50 CA86355). 7.4 Lecture series The program will have a monthly lecture series with alternating topics covering the three focus areas: nanotechnology, in vivo targeting/testing and oncology. The lectures will be given by scientists and engineers involved in the CCNE. In addition postdoctoral fellows will periodically present progress of individual projects. We will also invite outside guest speakers as part of our lecture series. 7.5 Annual retreat We plan an annual off-campus retreat to further foster interactions among CCNE members. Our experience has been that such retreats provide an outstanding opportunity for cross fertilization of ideas and for establishing new, formal collaborations. Although many of the Departments and Centers at MIT and Harvard, including the he MIT CCR and the DFHCC, hold such retreats, we feel that it is important to have a specific retreat for the faculty and trainees of the CCNE. Retreats will include platform and poster presentations as well as special lectures and workshops. We anticipate inviting the members of our internal and external advisory boards to attend the retreat. 7.5 Exchange program A major aspect of our educational program is its multidisciplinary training of students, postdoctoral fellows and staff. Formal mechanisms are in place to assign students and fellows to interdisciplinary joint programs (e.g. projects 2, 4, 5) through a central mechanism. We also encourage and facilitate student exchanges between programs. Several of our consortium members also have official exchange programs (visiting professors, sabbaticals, student exchanges, visitor programs) with other Universities and Programs. These programs are set up to share facilities, carry out collaborative research and cross-train oncology, chemistry, physics and engineering students. 7.6 Outreach to elementary schools: NanoSleuths This is a new project targeted at Cambridge inner city elementary school students. Dr. Angela Belcher has initiated this program to get students in local schools excited about nanoscience. This program has three major goals 1) to get students think about the nanoworld 2) to help increase the science infrastructure in local elementary schools 3) engage MIT undergraduate and graduate students in outreach to local schools. The NanoSleuths program will prepare special modules and teach applications of nanotechnology to cancer. Specifically, the NanoSleuths program will focus on modules for students to work in teams to solve "cases" that involve nano mysteries. MIT students and Dr. Belcher will help facilitate the cases. The cases to be investigated will be 1) "The Case of the Racing Slimes" 2) "The Case of the Slippery Atom" 3) The Case of the Stolen Gene 4) The Case of the Disappearing Light and 5) Nanorobots in cancer vessels, among others. 8. INTERACTIONS, OUTREACH 8.1 Interactions with Cancer Centers This CCNE is focused exclusively on the application of nanotechnology to problems in cancer diagnosis, treatment and disease monitoring, as this is reinforced by the tight integration with two NCI-designated Cancer Centers. The CCNE will be organized and administered by the MIT CCR (Dr. Jacks, Director), and will interact closely with the DFHCC (see letter from Dr. Livingston, Deputy Director). In addition, there multitude of Specialized Program of Research Excellence (SPORE) which are involved in the Projects. Projects 1 and 3 interact closely with the Prostate Spore at DFHCC lead by Dr. Kantoff. Drs. Kantoff and Rubin have played a pivotal role in the design of the current projects. They have also collaborated with Drs. Langer, Farokhzad, Josephson and Weissleder over a number of years. Project 2 will interact with research groups at the MIT CCR in developing siRNA-based targeting strategies for lung and brain tumor models under development. Through Dr. Jacks, Project 2 will interact with the DF/HCC lung cancer SPORE. Project 4 will rely on the Mouse Models Core, MIT CCR collaborators in the Jacks and Housman laboratory and the Ovarian Cancer group at MGH (Seiden, Chabner). Drs Weissleder and Seiden are corecipients of a Doris Duke award on ovarian cancer and have collaborated on developing methods for early detection and relapse of ovarian cancer in patients. Project 5 also relies on the Mouse Models Core and interactions with the clinical gastrointestinal research groups at DFHCC of which Dr. Weissleder is a member. Drs. Weissleder, Bhatia, Farokhzad and Ramaswamy are practicing physicians with appointments at MGH and/or BWH. Dr. Phillip Kantoff (collaborator of project 1 and 3) is Director of GU Oncology at DF/HCC and director of a GU Spore. Dr. Bruce Chabner (collaborator of project 3) is the Clinical Director of the MGH Cancer Center and Director of the "Phase 1-group" at DFHCC. Dr. Michael Seiden serves as Chairman of the MGH Cancer Center Clinical Protocol Committee, as well as Chairman of the Dana Farber-Harvard Gynecologic Oncology Research Committee. Dr. Daniel Haber is the Director of the MGH Cancer Center and Dr. David Livingston is the Deputy Director of the DFHCC. 8.2 Industrial outreach program (IOP) The consortium will establish a semi-annual meeting with industry leaders with joint sponsorship from MIT and Harvard Departments. The IOP is aimed at a) facilitating the rapid translation of most promising nanotechnology developments, b) strengthening external collaborations by facilitating mutually beneficial relationships and c) reviewing internal projects. The program will invite leading scientists from industry and our network of local laboratories to participate. The workshop will including talks and poster sessions on research, educational programs, shared experimental facilities and outreach activities. Based on our collective experience from the past several years the following large companies (among others) will be invited to participate in this Program: Merck, Novartis, Lilly, Abbot, Aventis, Glaxo, BMS, Johnson and Johnson, Proctor and Gamble, Genentech, Biogen, Analog Devices, Nanosys, Quantum Dot Corporation, General Electric and Siemens (see attached letters of interest from invitations already extended). 8.3 Clinical translation Members of this consortium have considerable expertise in clinical medicine, drug development, phase 1 clinical trials and translational research. Collectively, the team has developed the following materials for clinical use and/or has participated in clinical trials: [unreadable] Gliadel wafer, SonoPrep and others (76, 109) [unreadable] First clinical trials of magnetic nanoparticles (106) [unreadable] Development (119) and first clinical trials of lymphotropic magnetic nanoparticles (43, 44) [unreadable] Development and clinical testing of novel synthetic graft copolymers (17).

As stated above, the Project Manager, Shannon Cozzo, will oversee day-to-day management ofthe program. She currently serves in this role as a conduit between the PI/PD, Project Leaders and the centralized Koch Institute staff. Ms. Cozzo is involved in coordinating activities among affiliated institutions ofthe CCNE. These different units have separate and somewhat distinct financial management structures that will affect management ofthe various child and facility account that comprises the CCNE. The Project Manager is also responsible for coordinating all CCNE Alliance-related activities, steering committees, advisory boards and joint group meetings, monthly seminar series and the Education, Training and Outreach units, which are under the umbrella ofthe Administrative Core. She is located in the Koch Institute. This arrangement will ensure efficient coordination of all operations including management of finances, regulatory compliance and human resource support.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (14) Stem Cells and specific Challenge Topic, 14-DE-103: Enhancing Human Embryonic Stem (ES) Cell Culture Systems. The overall objective of this proposal is to develop and optimize a feeder-free in vitro culture system with a controlled culture microenvironment for human pluripotent stem cell growth and directed differentiation. In particular, high-throughput approaches will be used to develop culture materials with optimized surface properties incorporating defined extracellular matrix (ECM) components that allow for accurate control of oxygen partial pressure of the cell surface (pO2cell) to provide more physiological conditions and allow strategies to better mimic normal development processes. The developing embryo is exposed in vivo to very low oxygen levels, and there is growing evidence that oxygen concentration, together with cell-protein interactions, are important factors in differentiation and proliferation. In typical culture systems, pO2cell is unknown because of gradients in the culture medium. Currently, there are no commercially available methods or equipment for culturing cells under known, hypoxic conditions. Membranes of silicone rubber, which have very high oxygen permeability, will be used as a culture substrate so that cells are exposed to the same oxygen level as in the gas phase. Silicone rubber surfaces will be investigated in various forms: (1) unmodified, (2) chemically modified and functionalized with synthetic polymers, and (3) chemically modified with synthetic polymers to which ECM components are attached by physical adsorption or covalent linkage, including defined proteins and mixtures thereof. Cell compatible, ECM protein microarrays on functionalized silicone rubber will be created with a high-throughput microarray platform that we previously developed for culture of cells in order to screen a large number of cell-ECM-biomaterial interactions. The various cell substrates will be examined for cellular attachment, proliferation, and gene expression patterns at various levels of pO2cell using combinatorial techniques. Confirmatory experiments in macro-scale culture using culture vessels will be carried out with the most promising combinations. Similar experiments with the high-throughput microarray platform and culture vessels will be carried out for differentiation of human pluripotent stem cells to cardiomyocytes, which has been shown to benefit from hypoxic culture, as a model system. Accomplishment of the goal of the proposed studies will lead to elimination of conventional feeder layers and undefined xenogenic proteins and to the development and validation of a more well-defined and physiological culture platform for simultaneous variation and study of soluble factors, ECM-cell interactions, and pO2cell, thereby enabling enhancement of our understanding of the role of these factors in maintenance and differentiation of human pluripotent stem cells. This novel, generally applicable platform, will upgrade the capability and quantity of research in this field. Pluripotent stem cells hold enormous promise for drug screening, in vitro modeling of genetic disorders, and cell therapies and the proposed research will have wide impact on human health. At its most basic level, this research will provide information about the interaction of biomaterials, ECM components, and undifferentiated or differentiating human pluripotent stem cells at different known values of pO2cell that mimic physiological conditions, data for which has not previously been available except under normoxic culture. This information will advance our knowledge in the fields of developmental and stem cell biology as well as human pluripotent stem cell technology. In particular, we expect to learn how the choice of ECM proteins and pO2cell levels interact to aid directed differentiation. From a broader standpoint, the proposed high-throughput platform and macro-scale culture vessels that derive therefrom are tools that constitute enabling technology to culture human pluripotent stem cells under defined physiological conditions. They will improve the research infrastructure in terms of the capability for culturing human pluripotent stem cells. The knowledge acquired and tools developed may have profound effects on a wide variety of applications in many fields of human health. This will accelerate developments in applications such as drug screening, in vitro models of genetic disease, therapeutic replacement of diseased cells in major diseases such as heart disease, diabetes, and neural diseases such as Parkinson's, which annually affect millions of people in the United States. Indeed, differentiation of human pluripotent stem cells to cardiomyocytes, the proposed model system, is itself of high interest for generating cells and tissues for repair of cardiac tissues following ischemic heart disease. Furthermore, the culture vessels to be tested will be useful in culturing cardiac tissue. This collaborative study will integrate recent progress in the fields of bioengineering, biomaterials, and developmental biology to design a new culture platform and protocols that will facilitate cell-based therapies for treatment of a multitude of human diseases and other ES cell applications. PUBLIC HEALTH RELEVANCE: This project concerns improved methods to work with human stem cells so that they can develop into medically useful cells and tissues. For example, this can lead to ways to grow functioning heart muscle cells that can be used to treat heart disease.

The central aim of this CCNE project is to develop nanotechnologies for targeted combination pharmacotherapy using existing compounds with suboptimal pharmaceutical properties. The genomic revolution has resulted in the identification of approximately ~320 molecular targets and attempts to therapeutically utilize many of these have faced considerable development challenges (1, 2). More recently, advances in systems biology have aided in identifying synergistic pathways among these newly identified targets that may be concurrently utilized for more effective treatment of cancers. The development of nanotechnologies for effective delivery of multiple drugs or drug candidates in a temporally regulated manner to cancer cells can potentially overcome the development challenges faced to date, and result in harnessing the maximal benefits of cancer genomics and systems biology (3, 4). Our early work supported by the MIT-Harvard CCNE focused on engineering targeted nanoparticles for delivery of a single chemotherapeutic agent (docetaxel) for prostate cancer (PCa) therapy. Using a combinatorial process for engineering libraries of targeted nanoparticles by selfassembly, which is reproducible, we screened and Identified particles with optimal biophysicochemical properties. Particles with optimal properties are now in clinical development and approaching an IND in 2010./n the context of this proposal we hypothesize that 1) by engineering and blending distinct drugfunctionalized and ligand-functionalized polymers, with or without encapsulation of additional free drug molecules, we will be able to reproducibly engineer and characterize nanoparticles capable of delivering 2 or more drugs; and 2) by targeting these drug loaded nanoparticles to cancer cells we can achieve synergistic drug effects which may translate to better efficacy and tolerability making them suitable for potential clinical development. Herein we propose to develop technologies for co-delivery of up to 3 distinct anticancer agents for targeted combination chemotherapy. As a model cancer, building on our previous efforts, we propose to develop long circulating drug-conjugated targeted nanoparticles for differential uptake by PCa cells. We will aim to develop targeted nanoparticles with up to 3 distinct anticancer agents and place one candidate formulation on a development path toward an IND submission in 2014, setting the stage for clinical validation of our TempoSpatially-controlled Combination Chemotherapy (TSCC) platform in patients with hormone refractory prostate cancer (HRPC).

MIT and Harvard have excepfionally strong research and education programs in the biological/medical sciences on the one hand and in the physical/engineering sciences on the other, as well as strong programs in nanotechnology. There is a large pool of excellent students and MIT already has administrative and educational mechanisms in place for encouraging interdisciplinary activities among these students and the respective faculty members. We have structured our program to leverage this advantageous situation.

DESCRIPTION (provided by applicant): High throughout technologies has already significantly revolutionized fields such as genomics, proteomics, and drug discovery and formulation. This technology can similarly revolutionize the development of biomaterials for tissue engineering applications. A fundamental component of this proposal is to apply and advance our fully automated, high throughput discovery methods to push hESC tissue engineering closer towards clinical applications. Two key remaining limitations of existing hESC and iPSC methods are 1) the low efficiency and long time associated with stem cell differentiation into functional cell types such as chondrocytes and osteoblasts, and 2) suboptimal performance (e.g. mechanical properties, biocompatibility) of existing degradable materials used for tissue engineering. As such, we propose to develop both high throughput strategies to rapidly optimize the production of homogenous populations of key cell populations and degradable biomaterials that provide for improved cellular performance and low inflammation. Accordingly our specific aims are: Aim1. Develop efficient, rapid methods to differentiate human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into homogenous populations of craniofacial cells. We will use high-throughput approaches on hESC and iPSC cells to identify the optimal combinations of soluble (growth factor and small molecules), and insoluble factors (synthetic polymer surfaces) capable of committing ES cells to craniofacial precursor cells and fully committed osteogenic and chondrogenic cells. Aim 2: Develop biodegradable, non-inflammatory and mechanically appropriate 3D scaffold systems that can effectively deliver craniofacial cells to the site of injury. High-throughput libraries of hyaluronic acid (cartilage) and poly(2-amino ester) (bone) polymers will be synthesized and assessed for ability to form gels or solid porous scaffolds, respectively. Favorable materials will then be evaluated for either chondrocyte or bone cell compatibility. Aim 3: Assess performance of tissue engineered constructs developed in Aim 1 and 2 to generate cartilage and bone in vivo. Small animal model will be used to test osteogenesis in a cranial critical sized defect, while chondrogenesis will be evaluated subcutaneously. PUBLIC HEALTH RELEVANCE: We believe that this technology can similarly revolutionize the development of biomaterials for tissue engineering applications. A fundamental component of this proposal is to apply and advance our fully automated, high throughput discovery methods to push hESC and iPSC tissue engineering closer towards craniofacial clinical applications.

Project Summary/Abstract Our long-term goal is the development of systems providing controlled drug delivery of a broad set of therapeutics including those that are limited to parenteral routes. In this proposal, we build on our prior work to focus on the gastrointestinal (GI) barrier and specifically propose a platform enabling the high-throughput GI transport evaluation of novel formulations rapidly. Developing therapies which are compatible with oral administration, requires significant formulation and in vitro and in vivo evaluation for maximal oral bioavailability in humans. Current in vitro models of GI absorption are limited by their throughput and approximation of the physiologic state. Consequently, we propose: 1) the development of systems enabling prolonged culturing of intact mammalian GI tissue coupled to 2) high-throughput robotics to transform formulation development and study of the GI tract. These investigations are supported by strong preliminary data demonstrating: 1) culture conditions which maintain the presence of a broad set of cellular markers and drug transporters ex vivo in porcine GI tissue, 2) fabrication of prototype systems enabling high-throughput interrogation, 3) demonstration of predictive capacity of drug absorption for a large panel of drugs, 4) demonstration of near order of magnitude enhancement of uptake of a model molecule following a large-scale excipient screen. Currently, promising therapeutics which are poorly absorbed through the oral route can manifest in drug development delays on the order of years and many times no formulation solution is identified. The proposed work will target a critical unmet clinical need by providing tools to rapidly identify formulations that enable: maximal drug solubility and absorption and minimal local toxicity. Moreover, the proposed system will enable interrogation and study of the GI tract entero-endocrine system enabling the discovery of new therapies for metabolic disorders. Through this proposal we will develop a novel set of formulations and novel material-drug combinations enabling the oral delivery of drugs previously restricted to parenteral routes. Moreover, we will develop novel modulators of the enteroendocrine system providing novel solutions to the metabolic disease epidemic. In sum, this proposal aims to provide a platform akin to an ?intestine-on-a-chip? with the capacity to transform formulation science and the study of the GI tract with the potential to transform treatments for metabolic disease and other diseases of the GI tract.