Therapeutic Delivery Vehicles Research Articles

Nanoparticle-Based Antimicrobials: Surface Functionality is Critical.

Bacterial infections cause 300 million cases of severe illness each year worldwide. Rapidly accelerating drug resistance further exacerbates this threat to human health. While dispersed (planktonic) bacteria represent a therapeutic challenge, bacterial biofilms present major hurdles for both diagnosis and treatment. Nanoparticles have emerged recently as tools for fighting drug-resistant planktonic bacteria and biofilms. In this review, we present the use of nanoparticles as active antimicrobial agents and drug delivery vehicles for antibacterial therapeutics. We further focus on how surface functionality of nanomaterials can be used to target both planktonic bacteria and biofilms.

Therapeutic Use of 3β-[N-(N',N'-Dimethylaminoethane) Carbamoyl] Cholesterol-Modified PLGA Nanospheres as Gene Delivery Vehicles for Spinal Cord Injury.

Gene delivery holds therapeutic promise for the treatment of neurological diseases and spinal cord injury. Although several studies have investigated the use of non-viral vectors, such as polyethylenimine (PEI), their clinical value is limited by their cytotoxicity. Recently, biodegradable poly (lactide-co-glycolide) (PLGA) nanospheres have been explored as non-viral vectors. Here, we show that modification of PLGA nanospheres with 3β-[N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) enhances gene transfection efficiency. PLGA/DC-Chol nanospheres encapsulating DNA were prepared using a double emulsion-solvent evaporation method. PLGA/DC-Chol nanospheres were less cytotoxic than PEI both in vitro and in vivo. DC-Chol modification improved the uptake of nanospheres, thereby increasing their transfection efficiency in mouse neural stem cells in vitro and rat spinal cord in vivo. Also, transgene expression induced by PLGA nanospheres was higher and longer-lasting than that induced by PEI. In a rat model of spinal cord injury, PLGA/DC-Chol nanospheres loaded with vascular endothelial growth factor gene increased angiogenesis at the injury site, improved tissue regeneration, and resulted in better recovery of locomotor function. These results suggest that DC-Chol-modified PLGA nanospheres could serve as therapeutic gene delivery vehicles for spinal cord injury.

Protocells: Modular Mesoporous Silica Nanoparticle-Supported Lipid Bilayers for Drug Delivery.

Mesoporous silica nanoparticle-supported lipid bilayers, termed 'protocells,' represent a potentially transformative class of therapeutic and theranostic delivery vehicle. The field of targeted drug delivery poses considerable challenges that cannot be addressed with a single 'magic bullet'. Consequently, the protocell has been designed as a modular platform composed of interchangeable biocompatible components. The mesoporous silica core has variable size and shape to direct biodistribution and a controlled pore size and surface chemistry to accommodate diverse cargo. The encapsulating supported lipid bilayer can be modified with targeting and trafficking ligands as well as polyethylene glycol (PEG) to effect selective binding, endosomal escape of cargo, drug efflux prevention, and potent therapeutic delivery, while maintaining in vivo colloidal stability. This review describes the individual components of the platform, including the mesoporous silica nanoparticle core and supported lipid bilayer, their assembly (by multiple techniques) into a protocell, and the combined, often synergistic, performance of the protocell based on in vitro and in vivo studies, including the assessment of biocompatibility and toxicity. In closing, the many emerging variations of the protocell theme and the future directions for protocell research are commented on.

Chemokine CCL15 Mediates Migration of Human Bone Marrow-Derived Mesenchymal Stem Cells Toward Hepatocellular Carcinoma.

Mesenchymal stem cells (MSCs) possess the ability to migrate toward tumor sites and are regarded as promising gene delivery vehicles for cancer therapeutics. However, the factors that mediate this tropism have yet to be completely elucidated. In this study, through cytokine array analysis, chemokine CCL15 was found to be the most abundant protein differentially expressed in hepatocellular carcinoma (HCC) cell lines compared with a normal liver cell line. Serum CCL15 levels in HCC patients determined by enzyme linked immunosorbent assay were shown to be profoundly elevated compared with healthy controls. Immunohistochemical analysis indicated that CCL15 expression was much stronger in HCC tumor tissues than in adjacent nontumor tissues. Transwell migration assay suggested that CCL15 may be involved in chemotaxis of human MSCs (hMSCs) toward HCC in vitro and that this chemotactic effect of CCL15 is mediated via CCR1 receptors on hMSCs. Orthotopic animal models of HCC were established to investigate the role of CCL15 in hMSCs migration toward HCC in vivo. Both histological and flow cytometric analysis showed that significantly fewer hMSCs localized within 97H-CCL15-shRNA xenografts compared with 97H-green fluorescent protein xenografts after intravenous delivery. Finally, the possible effects of hMSCs on HCC tumor growth were also evaluated. Coculture experiments showed that hMSCs had no apparent effect on the proliferation of HCC cells in vitro In addition, systemic administration of hMSCs did not affect HCC tumor progression in vivo. Our data in this study help to elucidate the mechanism underlying the homing capacity of hMSCs toward HCC.

Investigation of Content, Stoichiometry and Transfer of miRNA from Human Neural Stem Cell Line Derived Exosomes.

Exosomes are small (30–100 nm) membrane vesicles secreted by a variety of cell types and only recently have emerged as a new avenue for cell-to-cell communication. They are natural shuttles of RNA and protein cargo, making them attractive as potential therapeutic delivery vehicles. MicroRNAs (miRNAs) are short non-coding RNAs which regulate biological processes and can be found in exosomes. Here we characterized the miRNA contents of exosomes derived from human neural stem cells (hNSCs). Our investigated hNSC line is a clonal, conditionally immortalized cell line, compliant with good manufacturing practice (GMP), and in clinical trials for stroke and critical limb ischemia in the UK (clinicaltrials.gov: NCT01151124, NCT02117635, and NCT01916369). By using next generation sequencing (NGS) technology we identified the presence of a variety of miRNAs in both exosomal and cellular preparations. Many of these miRNAs were enriched in exosomes indicating that cells specifically sort them for extracellular release. Although exosomes have been proven to contain miRNAs, the copy number quantification per exosome of a given miRNA remains unclear. Herein we quantified by real-time PCR a highly shuttled exosomal miRNA subtype (hsa-miR-1246) in order to assess its stoichiometry per exosome. Furthermore, we utilized an in vitro system to confirm its functional transfer by measuring the reduction in luciferase expression using a 3’ untranslated region dual luciferase reporter assay. In summary, NGS analysis allowed the identification of a unique set of hNSC derived exosomal miRNAs. Stoichiometry and functional transfer analysis of one of the most abundant identified miRNA, hsa-miR-1246, were measured to support biological relevance of exosomal miRNA delivery.

Human Mesenchymal Stem Cells in Cancer Therapy.

Human mesenchymal stem cells (hMSCs) have the ability to differentiate into several tissue types. Their use in cancer therapeutics or as therapeutic delivery vehicles has significant potential, particularly in their exosome/microvesicle-mediated tissue regeneration abilities. In this review, the potential use of hMSCs in cancer therapy is discussed.

Polyanhydrides: Synthesis, Properties, and Applications

This review focusses on polyanhydrides, a fascinating class of degradable polymers that have been used in and investigated for many bio-related applications because of their degradability and capacity to undergo surface erosion. This latter phenomenon is driven by hydrolysis of the anhydride moieties at the surface and high hydrophobicity of the polymer such that degradation and mass loss (erosion) occur before water can penetrate deep within the bulk of the polymer. As such, when surface-eroding polymers are used as therapeutic delivery vehicles, the rate of delivery is often controlled by the rate of polymer erosion, providing predictable and controlled release rates that are often zero-order. These desirable attributes are heavily influenced by polymer composition and morphology, and therefore also monomer structure and polymerization method. This review examines approaches for polyanhydride synthesis, discusses their general thermomechanical properties, surveys their hydrolysis and degradation processes along with their biocompatibility, and looks at recent developments and uses of polyanhydrides in drug delivery, stimuli-responsive materials, and novel nanotechnologies.

Mesenchymal Stem Cells Loaded with p5, Derived from CDK5 Activator p35, Inhibit Calcium-Induced CDK5 Activation in Endothelial Cells.

The potential use of stem cells as therapeutics in disease has gained momentum over the last few years and recently phase-I clinical trials have shown favourable results in treatment of a small cohort of acute stroke patients. Similarly, they have been used in preclinical models drug-loaded for the effective treatment of solid tumours. Here we have characterized uptake and release of a novel p5-cyclin-dependent kinase 5 (CDK5) inhibitory peptide by mesenchymal stem cells and showed release levels capable of blocking aberrant cyclin-dependent kinase 5 (CDK5) signaling pathways, through phosphorylation of cyclin-dependent kinase 5 (CDK5) and p53. These pathways represent the major acute mechanism stimulating apoptosis after stroke and hence its modulation could benefit patient recovery. This work indicates a potential use for drug-loaded stem cells as delivery vehicles for stroke therapeutics and in addition as anticancer receptacles particularly, if a targeting and/or holding mechanism can be defined.

Role of Four Different Kinds of Polyethylenimines (PEIs) in Preparation of Polymeric Lipid Nanoparticles and Their Anticancer Activity Study.

A series of polyethylenimines-coated poly(d,l-lactide-co-glycolide)/lipid nanoparticles (PPLs) were fabricated for delivering paclitaxel via a simple nano-precipitation method. Four kinds of polyethylenimines (PEIs) (800 Da-, 2000 Da- and 25 kDa-branched PEIs, and 25 kDa-linear PEI) were selected as a polymeric coating for the nanoparticles. The PPLs were evaluated for their cytotoxic effects towards tumor cells. The nanoparticles were uniform spheres with particle sizes ranging from 135.8 to 535.9 nm and zeta potentials between 13.5 and 45.4 mV. The content of lipids and PEIs were optimized at lipids content from 0 to 40% and PEI content from 2.5% to 10%, respectively. At 20% lipid content and 5% PEI content, the formulation was found to be optimal. In vitro experiments showed that 25 kDa-branched PEI coated PLGA/lipid nanoparticles (25k-bPPLs) had higher cytotoxicity than other PPLs in several cancer cell lines. Meanwhile, 25k-bPPLs maintained high cellular delivery efficiency without excessive toxicity, which was confirmed by confocal microscopy and flow cytometry analyses. Furthermore, 25k-bPPLs displayed excellent colloidal stability in pH 7.4 PBS. In conclusion, 25k-bPPLs are promising drug delivery vehicles for cancer therapeutics.

Micellar delivery of paclitaxel using a glucosamine-based glycopolymer for the treatment of prostate cancer

Event Abstract Back to Event Micellar delivery of paclitaxel using a glucosamine-based glycopolymer for the treatment of prostate cancer Alice Du1, Hongxu Lu2 and Martina H. Stenzel2 1 University of New South Wales, Centre for Advanced Macromolecular Design, School of Chemical Engineering, Australia 2 University of New South Wales, Centre for Advanced Macromolecular Design, School of Chemistry, Australia Introduction: The use of carbohydrates in the synthesis of polymeric nanoparticles has garnered interest in recent years, especially for its potential use as a therapeutic delivery vehicle for cancer treatment. For example, recent work reported by our group utilised the natural targeting ability of fructose decorated micelles for the treatment of triple-negative breast cancer[1]. Another carbohydrate of note is chitosan, a glucosamine-based glycopolymer which has shown potential in oral delivery approaches. However, one of the major disadvantages of chitosan lies in the handling difficulty of the macrostructure and control over its molecular weight. Hence, it is the aim of this study to synthesize a glucosamine monomer which is capable of undergoing controlled polymerization. Subsequent drug loading, micelle formation and in vitro studies will be used to evaluate the efficacy of the glycopolymer micellar system as a delivery vehicle for the treatment of prostate cancer. Materials and Methods: The solubility of glucosamine in organic solvents was increased through protection of its hydroxyl and amino functional groups. 2-hydroxyethyl acrylate was used to introduce alkene functionality to afford the protected glucosamine monomer (GluHEA). The polymerisation of GluHEA was controlled using a reversible addition-fragmentation chain transfer (RAFT) agent and subsequent chain extension was completed using 2-carboxyethyl acrylate (CEA). Paclitaxel was chemically conjugated to the CEA block and used to introduce hydrophobicity to the block. Deprotection of the glucosamine will then afford the hydrophilic block, thereby inducing micelle formation. In vitro biological studies will be conducted where the toxicity of the micelles will be evaluated against both monolayer cell culture as well as multicellular tumor spheroids. In addition, the migratory ability of the glucosamine-coated nanoparticles through Caco-2 (epithelial) cells will be investigated and compared to chitosan. Results: Synthesis of PGluHEA26-b-PCEA64 was successfully completed and controlled using 3-(benzylsulfanylthiocarbonyl sulfanyl)-propionic acid (BSPA) as the RAFT chain transfer agent (polydispersity ~1.3). Subsequent conjugation of paclitaxel to the carboxylic acid groups on the PCEA block was also successful. Conclusion: It is envisioned that the glucosamine coated micelles will offer a viable alternative to chitosan as an enhanced transcell delivery vehicle. In addition, the synthesis of a glucosamine monomer allows the structure of the block copolymer to be finely controlled, a characteristic not associated with naturally derived chitosan.

Polymer-based vehicles for therapeutic peptide delivery.

During the last decades increasing attention has been paid to peptides as potential therapeutics. However, clinical applications of peptide drugs suffer from susceptibility to degradation, rather short circulation half-life, limited ability to cross physiological barriers and potential immunogenicity. These challenges can be addressed by using polymeric materials as peptide delivery systems, owing to their versatile structures and properties. A number of polymer-based vehicles have been developed to stabilize the peptides and to control their release rates. Unfortunately, no single polymer or formulation strategy has been considered ideal for all types of peptide drugs. In this review, currently used and potential polymer-based systems for the peptide delivery will be discussed.

SiRNA-lipid nanoparticles with long-term storage stability facilitate potent gene-silencing in vivo

Considerable efforts have been directed towards discovering and developing delivery vehicles for RNA therapeutics. While most studies emphasize the efficacy and safety of these delivery vehicles, few reports conduct a comprehensive assessment of their storage stability, a critical property for practical applications. Here, we report a potent and safe lipid nanoparticle with long-term storage stability. Through chemical synthesis and screening of cationic lipids, a formulation has been identified that enables potent knockdown of hepatocyte proteins in mice upon intravenous administration (siRNA ED50 ~0.02mg/kg). Toxicity studies revealed that a dose of 2mg/kg was well tolerated in rats, the most sensitive rodent model. We identified that a cyclic chemical structure in cationic lipids improved particle stability. The nanoparticles showed over 1.5year storage stability as a liquid, with over 90% siRNA encapsulation without any changes in particle size. This novel delivery material has promising potential as a drug product that could bring RNA therapeutics to the treatment of liver-related disorders.

Evaluation of Polymeric Nanomedicines Targeted to PSMA: Effect of Ligand on Targeting Efficiency.

Targeted nanomedicines offer a strategy for greatly enhancing accumulation of a therapeutic within a specific tissue in animals. In this study, we report on the comparative targeting efficiency toward prostate-specific membrane antigen (PSMA) of a number of different ligands that are covalently attached by the same chemistry to a polymeric nanocarrier. The targeting ligands included a small molecule (glutamate urea), a peptide ligand, and a monoclonal antibody (J591). A hyperbranched polymer (HBP) was utilized as the nanocarrier and contained a fluorophore for tracking/analysis, whereas the pendant functional chain-ends provided a handle for ligand conjugation. Targeting efficiency of each ligand was assessed in vitro using flow cytometry and confocal microscopy to compare degree of binding and internalization of the HBPs by human prostate cancer (PCa) cell lines with different PSMA expression status (PC3-PIP (PSMA+) and PC3-FLU (PSMA-). The peptide ligand was further investigated in vivo, in which BALB/c nude mice bearing subcutaneous PC3-PIP and PC3-FLU PCa tumors were injected intravenously with the HBP-peptide conjugate and assessed by fluorescence imaging. Enhanced accumulation in the tumor tissue of PC3-PIP compared to PC3-FLU highlighted the applicability of this system as a future imaging and therapeutic delivery vehicle.

Nuclear Localization Signal-Enhanced Polyurethane-Short Branch Polyethylenimine-Mediated Delivery of Let-7a Inhibited Cancer Stem-Like Properties by Targeting the 3'-UTR of HMGA2 in Anaplastic Astrocytoma.

Anaplastic astrocytoma (AA) is a grade III glioma that often occurs in middle-aged patients and presents a uniformly poor prognosis. A small subpopulation of cancer stem cells (CSCs) possessing a self-renewing capacity is reported to be responsible for tumor recurrence and therapeutic resistance. An accumulating amount of microRNAs (miRNA) were found aberrantly expressed in human cancers and regulate CSCs. Efforts have been made to couple miRNAs with nonviral gene delivery approaches to target specific genes in cancer cells. However, the efficiency of delivery of miRNAs to AA-derived CSCs is still an applicability hurdle. The present study aimed to investigate the effectiveness and applicability of nonviral vector-mediated delivery of Let-7a with regard to eradication of AA and AA-derived CSC cells. Herein, our miRNA/mRNA microarray and RT-PCR analysis showed that the expression of Let-7a, a tumor-suppressive miRNA, is inversely correlated with the levels of HMGA2 and Sox2 in the AA side population (SP(+)) cells. Luciferase reporter assay showed that Let-7a directly targets the 3'-UTRs of HMGA2 in AA-SP(+) cells. Knockdown of HMGA2 significantly suppressed the protein expression of Sox2 in AA-SP(+) cells, whereas overexpression of HMGA2 upregulated Sox2 expression in AA-SP(-). Nuclear localization signal (NLS) peptides can facilitate nuclear targeting of DNA and are used to improve gene delivery. Using polyurethane-short branch polyethylenimine (PU-PEI) as a therapeutic delivery vehicle, we conjugated NLS with Let-7 and successfully delivered it to AA-SP(+) cells, resulting in significantly suppressed expression of HMGA2 and Sox2, tumorigenicity, and CSC-like abilities. This treatment facilitated the differentiation of AA-SP(+) cells into non-SP CSCs. Furthermore, PU-PEI-mediated delivery of NLS-conjugated Let-7a in AA-SP(+) cells suppressed the expression of drug-resistant and antiapoptotic genes, and increased cell sensitivity to radiation. Finally, the in vivo delivery of PU-PEI-NLS-Let-7a significantly suppressed the tumorigenesis of AA-SP(+) cells and synergistically improved the survival rate of orthotopically AA-SP(+)-transplanted immunocompromised mice when combined with radiotherapy. Therefore, PU-PEI-NLS-Let-7a is a potential novel therapeutic approach for AA.

Abstract 3669: Engineering the biointerface of synthetic high density lipoprotein nanoparticles enables efficient nucleic acid loading, delivery, and target gene regulation in cancer cells

Abstract Local treatment of cancer confined to the primary organ can be curative. However, other than a few success stories, locally advanced and metastatic cancer remains incurable. Ideally, new therapies are needed that specifically target cancer cells and derive efficacy by targeting molecular pathways that are unique to distinct tumor profiles and individual patients. Toward this end, we focus on the use of biomimicry to exploit mechanisms by which cancer cells uptake molecular cargo. Naturally, some cancer cells are dependent upon exogenous cholesterol for cellular signaling, growth, and in some cancers, such as prostate, steroidogenesis. However, cholesterol is a highly hydrophobic molecule that requires a delivery vehicle for systemic circulation and cellular delivery. High-density lipoproteins (HDLs) are natural nanostructures that engage cancer cells to deliver exogenous cholesterol. In addition to cholesterol, HDLs bind and deliver nucleic acids, such as microRNAs. Importantly, HDLs are targeted to a surface receptor, scavenger receptor type B-1 (SR-B1), whereupon binding allows for particle uptake and cargo delivery. SR-B1 has been shown to be overexpressed in human cancers, thus making HDLs a highly attractive delivery vehicle for therapeutic nucleic acid cargo. Because of the propensity of HDL to bind, stabilize, and deliver RNA our group has developed ways to synthesize lipoproteins nanoparticles and use them as therapeutic RNA delivery vehicles. Importantly, natural HDLs are highly dynamic species, which frequently undergo exchange of molecular surface components other lipoprotein species, as well as, HDLs. Recent work is focused on chemically tailoring the surface of synthetic HDL nanoparticles (HDL NPs) to understand RNA binding, stabilization, and cellular delivery. In addition, manipulating the surface chemical features of HDL also provides a way to better understand intermolecular exchange with native serum lipoproteins. Data demonstrate that by tailoring the surface chemistry of the HDL NP one can modulate the efficiency by which siRNAs can be incorporated onto the particle surface and delivered to target cells to reduce target gene expression in cancer cells. Further, data show the tendency for nanoparticle lipids to transfer to natural HDLs; however, siRNA formulated on the surface of the HDL NP remains bound, even when exposed to serum for prolonged periods. In short, exploration of HDL NPs for the systemic delivery of siRNAs may be a promising approach for targeted patient-specific cancer therapies. Citation Format: Kaylin M. McMahon, Don Vander Griend, Olga Volpert, C. Shad Thaxton. Engineering the biointerface of synthetic high density lipoprotein nanoparticles enables efficient nucleic acid loading, delivery, and target gene regulation in cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3669. doi:10.1158/1538-7445.AM2015-3669

Localization and Number of Au Nanoparticles in Optically Indexed Cells by FIB Tomography

Gold nanoparticles (GNPs) are gaining importance as therapeutic chemical delivery vehicles, medical diagnostic tools, and phototherapeutic and contrast enhancement agents. GNPs are uniquely suited for these biological uses because of their chemical stability, novel optical properties, and broad potential for functionalization. Additionally, each of these beneficial properties is further enhanced by the ability to manufacture GNPs in an almost endless combination of sizes and shapes. This versatility has allowed researchers to access and modify biological processes inside of a large variety of cells [1] and the observation of innocuous uptake of citrate stabilized GNPs [2]. To describe the effect of GNPs, characterization of affected cells and tissues is required from the macroscopic to nanoscopic level. In particular, the location of cells in the tissue or culture of interest and then the mapping of the number and spatial distribution of the GNPs inside of those cells is required, and frequently requires multiple imaging techniques [3]. We achieve the large scale mapping of mammalian stem cells using reflection optical microscopy and then explore the location and number of the nanoparticles inside these cells after exposure to 60 nm GNPs using focused ion beam – scanning electron (FIB-SEM) based tomography.

Capsosomes as Long-Term Delivery Vehicles for Protein Therapeutics.

We report the preparation of polymer capsules containing liposomal subcompartments, termed capsosomes, and their ability for the sustained delivery of protein therapeutics. Capsosomes were formed through the layer-by-layer (LbL) assembly of polymers and protein-loaded liposomes, followed by the formation of a capsule membrane based on disulfide cross-linked poly(methacrylic acid). The loading capacities of a model cargo (lysozyme) and brain-derived neurotrophic factor (BDNF), an important neurotrophin that has significant physiological functions on the nervous system, were determined, and the long-term release kinetics of the proteins was investigated in simulated physiological conditions. The capsosomes exhibited protein loading and release behavior that can be tuned by the lipid composition of the liposomal compartments, where inclusion of anionic lipids resulted in enhanced protein loading and slower release over the course of 80 days. These findings highlight the potential of capsosomes for the long-term delivery of protein therapeutics.

Quantitative proteomics and transcriptomics reveals metabolic differences in attracting and non-attracting human-in-mouse glioma stem cell xenografts and stromal cells

Bone marrow-derived human mesenchymal stem cells (BM-hMSCs) show promise as cell-based delivery vehicles for anti-glioma therapeutics, due to innate tropism for gliomas. However, in clinically relevant human-in-mouse glioma stem cell xenograft models, BM-hMSCs tropism is variable. We compared the proteomic profile of cancer and stromal cells in GSCXs that attract BM-hMSCs (“attractors”) with those to do not (“non-attractors”) to identify pathways that may modulate BM-hMSC homing, followed by targeted transcriptomics. The results provide the first link between fatty acid metabolism, glucose metabolism, ROS, and N-glycosylation patterns in attractors. Reciprocal expression of these pathways in the stromal cells suggests microenvironmental cross-talk.

Advances in Drug Delivery

Advances in Drug Delivery

329. Identifying Pharmacological Enhancers of Adenoviral Vector Replication and Transgene Expression

Adenovirus (Ad) vectors are currently the most commonly used vehicle for therapeutic gene delivery in human gene therapy, and oncolytic Ad-based gene therapy has shown promise in several types of cancer. We are interested in identifying compounds that enhance or inhibit Ad gene expression and replication.We have designed a novel Ad vector containing the RFP gene within the late transcription unit. Thus, the level of RFP expression correlates to the extent of virus replication. We have also validated a high-throughput system for screening small molecules using this vector. The anticancer chemotherapy drug vorinostat was initially expected to increase gene expression from the vector since it is a pan-histone deacetylase inhibitor (HDACI), and HDACIs have been shown to increase Ad transgene expression. However, our assays show that vorinostat significantly delays the onset of vector replication and transgene expression, and that these effects may be specific to the inhibition of class I HDACs. We are currently working on elucidating the mechanism by determining the role of specific class I HDACs and the downstream effects of HDAC-activity loss. Consequences of HDAC inhibition on vector replication and transgene expression will also be studied in vivo. In the future, we will use our Ad construct to screen small-molecule libraries and identify novel enhancers of these processes.