Michael H. Jones

Leishmaniasis is a protozoan disease responsible for significant morbidity and mortality. There is no recommended vaccine to protect against infection. In this study, transgenic Leishmania tarentolae expressing gamma glutamyl cysteine synthetase (γGCS) from three pathogenic species were produced and their ability to protect against infection determined using models of cutaneous and visceral leishmaniasis. The ability of IL-2-producing PODS^® to act as an adjuvant was also determined in L. donovani studies. Two doses of the live vaccine caused a significant reduction in L. major (p < 0.001) and L. donovani (p < 0.05) parasite burdens compared to their respective controls. In contrast, immunisation with wild type L. tarentolae, using the same immunisation protocol, had no effect on parasite burdens compared to infection controls. Joint treatment with IL-2-producing PODS^® enhanced the protective effect of the live vaccine in L. donovani studies. Protection was associated with a Th1 response in L. major and a mixed Th1/Th2 response in L. donovani, based on specific IgG1 and IgG2a antibody and cytokine production from in vitro proliferation assays using antigen-stimulated splenocytes. The results of this study provide further proof that γGCS should be considered a candidate vaccine for leishmaniasis.

Protein therapeutics are often compromised by sub-optimal biodistribution contributing to poor efficacy and adverse events. Drug delivery mechanisms better able to target protein drugs to the disease site and provide localized, sustained release have the potential to transform therapeutic standards. PODS ^® crystals (PODS) are natural-mimetic, micron-scale protein co-crystals engineered to incorporate a protein cargo that can be sustainably released under the action of resident proteases. PODS are efficiently taken up by phagocytic cells with the cargo protein subsequently released in a bioactive form. Since blood-circulating phagocytic cells, including monocytes, are actively recruited into diseased and inflamed tissue, such as the tumour microenvironment, we postulated that monocyte/macrophage-mediated PODS delivery could be used as a molecular “Trojan horse” to efficiently deliver therapeutic proteins to target cells. This could improve the pharmacodynamics and pharmacokinetics of protein drug delivery to treat systemic and disseminated diseases. Interleukin-2 (IL-2) is notoriously toxic at the high doses required for therapeutic efficacy. Here, we demonstrate the therapeutic efficacy and tolerability of low doses of PODS containing IL-2 cargo (PODS-IL-2) administered intravenously in a mouse model of melanoma. We further demonstrate the therapeutic benefit of PODS delivering IL-2, interleukin-15 (IL-15) and interferon gamma (IFN-γ) in a mouse model of renal cell carcinoma at two doses. Efficacy was seen in both doses with the higher dose generating rapid and complete rejection of the tumour in some of the mice treated with each cytokine. This study provides proof-of-concept for the utility of intravenously administered PODS to provide a generalised and widely applicable mechanism to effectively deliver protein drugs for the therapy of cancer and potentially other diseases.

Many cells possess the ability to engulf and incorporate particles by phagocytosis. This active process is characteristic of microorganisms as well as higher order species. In mammals, monocytes, macrophages and microglia are among so-called professional phagocytes. In addition, cells such as fibroblast and chondrocytes are classified as non-professional phagocytes. Professional phagocytes play important roles in both the innate and adaptive immune response, wound healing and tissue homeostasis. Consequently, these cells are increasingly studied as targets and vectors of therapeutic intervention to treat a range of diseases. Professional phagocytes are notoriously difficult to transfect limiting their study and manipulation. Consequently, efforts have shifted towards the development of nanoparticles to deliver a cargo to phagocytic cells via phagocytosis. However, this approach carries significant technical challenges, particularly for protein cargos. We have focused on the development of nanoscale co-crystalline protein depots, known as PODS®, that contain protein cargos, including cytokines. Here, we show that PODS are readily phagocytosed by non-professional as well as professional phagocytic cells and have attributes, such as highly sustained release of cargo, that suggest potential utility for the study and exploitation of phagocytic cells for drug delivery. Monocytes and macrophages that ingest PODS retain normal characteristics including a robust chemotactic response. Moreover, the PODS-cytokine cargo is secreted by the loaded cell at a level sufficient to modulate the behavior of surrounding non-phagocytic cells. The results presented here demonstrate the potential of PODS nanoparticles as a novel molecular tool for the study and manipulation of phagocytic cells and for the development of Trojan horse immunotherapy strategies to treat cancer and other diseases.

Many cells possess the ability to engulf and incorporate particles by phagocytosis. This active process is characteristic of microorganisms as well as higher order species. In mammals, monocytes, macrophages, and microglia are among the so-called professional phagocytes. In addition, cells such as fibroblast and chondrocytes are classified as nonprofessional phagocytes. Professional phagocytes play important roles in both the innate and adaptive immune responses, wound healing, and tissue homeostasis. Consequently, these cells are increasingly studied as targets and vectors of therapeutic intervention to treat a range of diseases. Professional phagocytes are notoriously difficult to transfect limiting their study and manipulation. Consequently, efforts have shifted towards the development of nanoparticles to deliver a cargo to phagocytic cells via phagocytosis. However, this approach carries significant technical challenges, particularly for protein cargos. We have focused on the development of nanoscale cocrystalline protein depots, known as PODS, that contain protein cargos, including cytokines. Here, we show that PODS are readily phagocytosed by nonprofessional as well as professional phagocytic cells and have attributes, such as highly sustained release of cargo, that suggest potential utility for the study and exploitation of phagocytic cells for drug delivery. Monocytes and macrophages that ingest PODS retain normal characteristics including a robust chemotactic response. Moreover, the PODS-cytokine cargo is secreted by the loaded cell at a level sufficient to modulate the behavior of surrounding nonphagocytic cells. The results presented here demonstrate the potential of PODS nanoparticles as a novel molecular tool for the study and manipulation of phagocytic cells and for the development of Trojan horse immunotherapy strategies to treat cancer and other diseases.

The spatial and temporal availability of cytokines, and the microenvironments this creates, is critical to tissue development and homeostasis. Creating concentration gradients in vitro using soluble proteins is challenging as they do not provide a self-sustainable source. To mimic the sustained cytokine secretion seen in vivo from the extracellular matrix (ECM), we encapsulated a cargo protein into insect virus-derived proteins to form nanoparticle co-crystals and studied the release of this cargo protein mediated by matrix metalloproteinase-2 (MMP-2) and MMP-8. Specifically, when nerve growth factor (NGF), a neurotrophin, was encapsulated into nanoparticles, its release was promoted by MMPs secreted by a PC12 neuronal cell line. When these NGF nanoparticles were spotted onto a cover slip to create a uniform circular field, movement and alignment of PC12 cells via their extended axons along the periphery of the NGF nanoparticle field was observed. Neural cell differentiation was confirmed by the expression of specific markers of tau, neurofilament, and GAP-43. Connections between the extended axons and the growth cones were also observed, and expression of connexin 43 was consistent with the formation of gap junctions. Extensions and connection of very fine filopodia occurred between growth cones. Our studies indicate that crystalline protein nanoparticles can be utilized to generate a highly stable cytokine gradient microenvironment that regulates the alignment and differentiation of nerve cells. This technique greatly simplifies the creation of protein concentration gradients and may lead to therapies for neuronal injuries and disease.

Despite recent efforts in discovering novel long non-coding RNAs (lncRNAs) and unveiling their functions in a wide range of biological processes their applications as biotechnological or therapeutic tools are still at their infancy. We have recently shown that AS Uchl1, a natural lncRNA antisense to the Parkinson’s disease-associated gene Ubiquitin carboxyl-terminal esterase L1 (Uchl1), is able to increase UchL1 protein synthesis at post-transcriptional level. Its activity requires two RNA elements: an embedded inverted SINEB2 sequence to increase translation and the overlapping region to target its sense mRNA. This functional organization is shared with several mouse lncRNAs antisense to protein coding genes. The potential use of AS Uchl1-derived lncRNAs as enhancers of target mRNA translation remains unexplored. Here we define AS Uchl1 as the representative member of a new functional class of natural and synthetic antisense lncRNAs that activate translation. We named this class of RNAs SINEUPs for their requirement of the inverted SINEB2 sequence to UP-regulate translation in a gene-specific manner. The overlapping region is indicated as the Binding Doman (BD) while the embedded inverted SINEB2 element is the Effector Domain (ED). By swapping BD, synthetic SINEUPs are designed targeting mRNAs of interest. SINEUPs function in an array of cell lines and can be efficiently directed towards N-terminally tagged proteins. Their biological activity is retained in a miniaturized version within the range of small RNAs length. Its modular structure was exploited to successfully design synthetic SINEUPs targeting endogenous Parkinson’s disease-associated DJ-1 and proved to be active in different neuronal cell lines. In summary, SINEUPs represent the first scalable tool to increase synthesis of proteins of interest. We propose SINEUPs as reagents for molecular biology experiments, in protein manufacturing as well as in therapy of haploinsufficiencies.

Current clinical and histopathological criteria used to define lung squamous cell carcinomas (SCCs) are insufficient to predict clinical outcome. To make a clinically useful classification by gene expression profiling, we used a 40 386 element cDNA microarray to analyse 48 SCC, nine adenocarcinoma, and 30 normal lung samples. Initial analysis by hierarchical clustering (HC) allowed division of SCCs into two distinct subclasses. An additional independent round of HC induced a similar partition and consensus clustering with the non-negative matrix factorization approach indicated the robustness of this classification. Kaplan–Meier analysis with the log-rank test pointed to a nonsignificant difference in survival (P=0.071), but the likelihood of survival to 6 years was significantly different between the two groups (40.5 vs 81.8%, P=0.014, Z-test). Biological process categories characteristic for each subclass were identified statistically and upregulation of cell-proliferation-related genes was evident in the subclass with poor prognosis. In the subclass with better survival, genes involved in differentiated intracellular functions, such as the MAPKKK cascade, ceramide metabolism, or regulation of transcription, were upregulated. This work represents an important step toward the identification of clinically useful classification for lung SCC.

The utility of cancer cell lines depends largely on their accurate classification, commonly based on histopathological diagnosis of the cancers from which they were derived. However, because cancer is often heterogeneous, the cell line, which also has the opportunity to alter in vitro , may not be representative. Yet without the overall architecture used in histopathological diagnosis of fresh samples, reclassification of cell lines has been difficult. Gene-expression profiling accurately reproduces histopathological classification and is readily applicable to cell lines. Here, we compare the gene-expression profiles of 41 cell lines with 44 tumors from lung cancer. These profiles were generated after hybridization of samples to four replicate 7,685-element cDNA microarrays. After removal of genes that were uniformly up- or down-regulated in fresh compared with cell-line samples, cluster analysis produced four major branch groups. Within these major branches, fresh tumor samples essentially clustered according to pathological type, and further subclusters were seen for both adenocarcinoma (AC) and small cell lung carcinoma (SCLC). Four of eight squamous cell carcinoma (SCC) cell lines clustered with fresh SCC, and 11 of 13 SCLC cell lines grouped with fresh SCLC. In contrast, although none of the 11 AC cell lines clustered with AC tumors, three clustered with SCC tumors and six with SCLC tumors. Although it is possible that preexisting SCC or SCLC cells are being selected from AC tumors after establishment of cell lines, we propose that, even in situ , AC will ultimately progress toward one of two poorly differentiated phenotypes with expression profiles resembling SCC or SCLC.