Genesegues (United States)

Passive topical administration of nucleic acid drugs and vaccines is a desirable goal for patient-friendly manipulation of target cells in the skin. However, two major delivery challenges that have impeded development are 1) penetrating the stratum corneum, and 2) avoiding endosomal entrapment and degradation. GeneSegues has developed a novel drug delivery technology, sub-50 nanometer (s50) crystallized capsules, for intact delivery of nucleic acids to intracellular sites in target cells. s50 capsules are directed to specific tissue and cells by formulating appropriate targeting proteins, peptides or antibody ligands as the crystallized capsule shell. The novel size and structure of s50 capsules allows for efficient penetration of the stratum corneum and, by co-opting the non-endosomal pathway referred to as “lipid rafts”, intact delivery of nucleic acid cargo to the nucleus and cytoplasm of the target cell. Here we show in pilot studies using pig dermis organ culture as an established and relevant model for human skin, transcutaneous passage of plasmid-bearing s50 capsules with cell-specific targeting. In these studies, we compared fibronectin, hyaluronan and tenascin as shell targeting components. We extended these studies of passive cutaneous delivery, using plasmid-bearing, fibronectin-coated s50 capsules, to in vivo timecourse studies in murine paws where stratum corneum layers in footpads are approximately 50 um thick, demonstrating uniform transfection of underlying basal keratinocytes. In a further extension, using hyaluronan-coated s50 capsules together with a novel adjuvant, we demonstrate a functional response to model antigen in studies of passive topical DNA vaccine adminstration in weanling pigs. Skin is a powerful immune organ, not conveniently addressed by current methods of vaccine administration, e.g., injection, gene gun, or electroporation. We found improvement in mucosal and cell-mediated immune responses, relative to intradermal protein antigen injection that was biased towards distal and draining nodal lymphoctyes, demonstrating a functional response in a clinically relevant model to topical administration of nucleic acids using s50 capsules. We expect that with further characterization, these studies may provide a solid framework for further development of s50-mediated cutaneous delivery of nucleic acid drugs and vaccines. Grant acknowledgments: NIH R43 CA119556, R43 AI60268

Current magnetic resonance (MR) contrast agents utilize vascular spaces to visualize and enhance neoplasms. The nanocapsule (sub-50 nanometer capsule) is a novel delivery technology capable of intracellular delivery of large cargos in a tissue- and cell-specific manner. These nanocapsules derive their specificity from a coating of ligands that have the potential to specifically target tumors. We are developing nanoencapsulated MR agents that can be delivered intracellularly to enhance MR imaging of localized and metastatic cancer. We prepared tenfibgen-coated ferrous oxide (FeO) nanoparticles and compared them to tenfibgen-coated ferrous oxide fluid by i) morphology (atomic force microscopy., ii) surface charge (zeta potential by dynamic light scattering) iii) incorporation (colorometric assay, ICP-AES), iv) cellular proliferation by thymidine incorporation and v) cellular uptake by iron histology. We found that although incorporation is low, nanocapsule targeting is effective enough to provide sufficient cellular accumulation for histology as well as functional imaging. We also showed 1000-fold improvement in 3D cell uptake studies with retargeted iron nanoparticles conducted on matrix-coated tissue engineering scaffold to reconstitute lipid rafts and caveolae required for cellular uptake. Mice bearing PC-3 xenograft tumors were treated with nanoencapsulated siRNA labeled with I-128 and measured acitivity via nuclear activation analysis. Tumor and injection site were primary sites of s50 tenfibgen siRNA-bearing capsule accumulation at 2 hours postinjection timepoint. Capsules also accumulated significantly in enlarged lymph nodes in the metastatic PC-3 model. No accumulation occurred in the organs of the reticular endothelial system. We then assessed the ability of MR to identify a signal change in mice bearing orthotopic PC-3 xenografts. The mice were treated with the nanoencapsulated FeO, 100 ng dose of FeO tenfibgen nanocapsules or a dose of about 3 ug/kg. The mice were then sacrificed and imaged. A decrease signal on T1 weighted MRI in contrasted tumor was detected suggesting FeO uptake. Perls+DAB staining demonstrated Fe staining in microvessels and tumor cells. This intratumoral distribution was consistent with reporter gene expression studies conducted utilizing the tenfibgen capsule. In conclusion, we have developed a nanoencapsulate FeO agent that is taken up intracellularly in tumors and can produce a detectable MR signal change. Future work will focus on encapsulating Ferric Dextran as well as candidate agents that can produce a positive signal change on MRI.

Purpose: To investigate targeting and efficacy of s50 capsules for delivery of biotherapeutics to primary and distant tumors by subcutaneous (SQ) injection. Methods s50 capsules comprised of a tumor-targeting Tenfibgen (TBG) shell and bearing either Dysprosium (TBGDy) or a mixture of RNAi oligonucleotides against Casein Kinase 2 (CK2) α and α′ kinase subunits (GS-10), were synthesized by dispersion atomization (Fig.1). Two metastatic head and neck cancer (HNC) xenograft models, FaDu and SCC-47, were flank-injected into nude mice to test s50 targeting and efficacy. Specific targeting was assayed by delivering TBGDy via IV or SQ administration and measuring biodistribution. Mice were dissected and Dy content was quantitated in blood and tissues by neutron activation analysis for the isotope Dy163m. Capsule localization in tissue was documented by direct far-red fluorescence of Dy and indirect immunofluorescence of a Fab fragment “spiked” into the capsule shell. GS-10 efficacy in xenograft models was assayed by measuring growth inhibition via primary tumor weights and CK2 mRNA transcript levels by qPCR. GS-10 efficacy on distant brain and lung tumors was determined by cytokeratin-14 (K-14) expression. Results: Biodistribution studies showed higher Dy concentration in tumor than liver for SQ routes (Fig. 2). Dy accumulation in primary tumors peaked at 48hrs post-SQ administration and was comparable to 4hrs post-IV administration (28% vs. 16% ID/g). Dy colocalizationn with capsule shell was confirmed by confocal microscopy supporting interpretation of Dy163m as capsule signal (Fig. 3). In the 30-day FaDu efficacy study, a 100 ug/kg daily (tumor-remote) SQ dose, decreased K-14, 47 and 79% in brains and lungs, respectively (n=5). In the 21-day SCC-47 model, significant growth inhibition of was observed in primary tumor weights relative to diluent and sugar capsule treatment respectively (92.8, 89.8%). CK2 RNA levels were reduced relative to sugar capsule-treated tumors (α, α′: 25.4%, 24.4%, n=3) while further anti-CK2 transcript activity by translational blockade, and CK2 protein downregulation was found by microscopy. Conclusion: Subcutaneous administration of s50-delivered RNAi is a viable strategy for treatment of metastatic cancer.

A high initial priming dose of the novel RNAi therapeutic GS-10 was investigated as a strategy to elevate important RISC complex proteins that have the potential to increase efficacy of subsequent RNAi treatment regimens in solid tumor cancers. GS-10 is a tumor-targeted 20 nm crystalline capsule bearing single-stranded RNAi oligos against Protein Kinase CK2 (CK2), and a ligand-coated shell derived from Tenascin-C (tenfibgen). Tenascin-C is upregulated in multiple solid tumors, and is a promising targeting ligand for tumors throughout their lifecycle. The ultrasmall capsule size enables efficient raft-mediated delivery of oligos to the perinuclear space of the target cell, and the ability to reach metastases without reliance on EPR. To date, strategies for RNAi-treatment of solid tumor cancers have been sub-optimal due to, at least in part, the varied levels of RNA degradation machinery across patients, tumors and tissues. For example, we have found wide variation in Ago2 protein levels, a key RISC component for RNA cleavage, in human head and neck cancer (HNC) tissues. Here we investigate a strategy for overcoming this limitation for an RNAi therapeutic in solid tumors. We have previously established that SQ doses of GS-10 at 10-100 ug/kg/day have antitumor efficacy, coincident with CK2 protein knockdown, in several mouse models. In an SCC-47 HNC xenograft model, tumor growth inhibition of 86% was observed after GS-10 treatment compared to controls, despite low baseline Ago2 levels. Interestingly, these tumors express relatively high Ago4 levels. FISH analysis of GS-10-treated SCC-47 tumors revealed numerous punctate structures containing mRNA CK2 transcript. Similarly, structures containing Ago4 and GW182 were observed by confocal microscopy, altogether suggesting these RISC proteins mediate transcript sequestration by forming GW-bodies, resulting in tumor inhibition. Now, we are evaluating a strategy using high-dose IV bolus injection as priming to elevate expression of multiple RISC proteins, including Ago2, Ago4 and GW182, in the SCC-47 model. A priming dose of GS-10 at 10 mg/kg showed a 4-fold increase in Ago2 levels in primary tumors vs. treatment controls at 72 hours post-injection. Similarly, a 2-fold increase in Ago4 and GW182 levels was also observed. To establish that GS-10 was reaching tumors in vivo to induce these changes, distribution of an isotopic tenfibgen surrogate of GS-10 to primary tumor and lymph node metastases was confirmed following IV bolus administration. Additional RISC protein and tumor efficacy data from a later timepoint is being analyzed and will be reported. In summary, these data suggest that high-dose IV priming with GS-10 can elevate multiple RISC proteins, setting tumors up for more effective response to subsequent low-dose treatment using this novel therapeutic candidate.

Head and neck cancer long term survival has only experienced marginal gains over the past 3 decades. Further, there is inadequate understanding of the biology of carcinogenesis and recurrence, as well as its relation to the microenvironment. Deeper understanding of these areas would provide improved molecular target identification. In pursuit of this goal, a small clinical trial collected lesion and adjacent normal-appearing mucosa for subsequent RNA-seq analysis. Patients were grouped by post-surgical pathology as either precancer (hyperplasia - severe dysplasia) or cancer (carcinoma in situ - early stage invasive cancer). Following identification of differentially expressed (DE) genes, DE genesets were submitted for Ingenuity Pathway Analysis (IPA). Hierarchical clustering illustrates distinct separation between lesion and perilesional normal mucosa of the top 100 DE genes. Among the top 25 dysregulated pathways, 50% were associated with creation of fibrotic tumor microenvironment (TME), 20% were related to changes in immune populations inhabiting the TME and 10% devoted to metabolism changes. Subgroup analysis, (precancer vs. cancer), revealed dysregulation of metabolism (~50%) predominating in precancer. Metabolism remained an important dysregulation at 30% of the top 25 pathways in cancer. Protein network analysis, (Metascape on-line tool), confirmed IPA results, illustrating an extensive, previously undescribed, interconnectedness of fibrosis with shifts in fatty acid metabolism from oxidative to gluconeogenesis, providing a foundation for choosing targets amenable to cancer prevention. Several notable pathways are likely contributed to by inflammatory and other cells in the milieu, not precancer cells themselves. So, we dove deeper, using EpIC (Epitope Immunogenicity Characterization) algorithm to assess relative percentages of non-tumor cells based on 20-count gene signatures. Gene expression favored a profile of significantly increasing cancer-associated fibroblasts, decreasing CD-8 killer T cells, and increasing vascular endothelial cells during progression, with macrophage content slightly increasing in cancer specimens. These findings suggest interaction between immunoinflammatory milieu and precancerous cells promoting malignancy. Several high yield target pathways are related to published mechanisms of action for drugs of high interest to our cancer prevention program (pioglitazone/metformin). Further, we confirmed protein network analysis in an additional oral carcinoma dataset from Conway et. al, (Oncotarget 2015; doi: 10.18632/oncotarget.5529). Abstract 2590

CK2 is a highly conserved, ubiquitous, signal responsive protein serine/threonine kinase. CK2 promotes cell proliferation and suppresses apoptosis, and increased CK2 expression is observed in all cancers examined. We previously reported that direct injection of antisense (AS) CK2α phosphorothioate oligonucleotides (PTO) into xenograft prostate tumors in mice significantly reduced tumor size. Downregulation of CK2α in tumor cells in vivo appeared to result in overexpression of CK2α' protein. This suggested that in cancer cells downregulation of CK2α might be compensated by CK2α' in vivo, prompting us to design a bispecific (bs) AS PTO (bs-AS-CK2) targeting both catalytic subunits. bs-AS-CK2 reduced CK2α and α' protein expression, decreased cell proliferation, and induced apoptosis in cultured cells. Biodistribution studies of administered bs-AS-CK2 oligonucleotide demonstrated its presence in orthotopic prostate xenograft tumors. High dose injections of bs-AS-CK2 resulted in no damage to normal liver or prostate, but induced extensive cell death in tumor tissue. Intraperitoneal treatment with bs-AS-CK2 PTO decreased orthotopic tumor size and downregulated both CK2 mRNA and protein expression. Tumor reduction was accomplished using remarkably low doses and was improved by dividing the dose using a multi-day schedule. Decreased expression of the key signaling pathway proteins NF-κB p65 and AKT was also observed. We propose that the molecular downregulation of CK2 through bispecific targeting of the two catalytic subunits may be uniquely useful for therapeutic elimination of tumors.