Preclinical Enzyme Replacement Therapy with a Recombinant ß-Galactosidase-Lectin Fusion for CNS Delivery and Treatment of GM1-Gangliosidosis.

GM1-gangliosidosis is a catastrophic, neurodegenerative lysosomal storage disease caused by a deficiency of lysosomal ß-galactosidase (ß-Gal). The primary substrate of the enzyme is GM1-ganglioside (GM1), a sialylated glycosphingolipid abundant in nervous tissue. Patients with GM1-gangliosidosis present with massive and progressive accumulation of GM1 in the central nervous system (CNS), which leads to mental and motor decline, progressive neurodegeneration, and early death. No therapy is currently available for this lysosomal storage disease. Here, we describe a proof-of-concept preclinical study toward the development of enzyme replacement therapy (ERT) for GM1-gangliosidosis using a recombinant murine ß-Gal fused to the plant lectin subunit B of ricin (mß-Gal:RTB). We show that long-term, bi-weekly systemic injection of mß-Gal:RTB in the ß-Gal-/- mouse model resulted in widespread internalization of the enzyme by cells of visceral organs, with consequent restoration of enzyme activity. Most importantly, ß-Gal activity was detected in several brain regions. This was accompanied by a reduction of accumulated GM1, reversal of neuroinflammation, and decrease in the apoptotic marker caspase 3. These results indicate that the RTB lectin delivery module enhances both the CNS-biodistribution pattern and the therapeutic efficacy of the ß-Gal ERT, with the potential to translate to a clinical setting for the treatment of GM1-gangliosidosis.

Targeting Macromolecules to CNS and Other Hard-to-Treat Organs Using Lectin-Mediated Delivery.

The greatest challenges for therapeutic efficacy of many macromolecular drugs that act on intracellular are delivery to key organs and tissues and delivery into cells and subcellular compartments. Transport of drugs into critical cells associated with disease, including those in organs protected by restrictive biological barriers such as central nervous system (CNS), bone, and eye remains a significant hurdle to drug efficacy and impacts commercial risk and incentives for drug development for many diseases. These limitations expose a significant need for the development of novel strategies for macromolecule delivery. RTB lectin is the non-toxic carbohydrate-binding subunit B of ricin toxin with high affinity for galactose/galactosamine-containing glycolipids and glycoproteins common on human cell surfaces. RTB mediates endocytic uptake into mammalian cells by multiple routes exploiting both adsorptive-mediated and receptor-mediated mechanisms. In vivo biodistribution studies in lysosomal storage disease models provide evidence for the theory that the RTB-lectin transports corrective doses of enzymes across the blood-brain barrier to treat CNS pathologies. These results encompass significant implications for protein-based therapeutic approaches to address lysosomal and other diseases having strong CNS involvement.

The RNA-binding protein HuR contributes to neuroinflammation by promoting C-C chemokine receptor 6 (CCR6) expression on Th17 cells.

In both multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), the C-C chemokine receptor 6 (CCR6) is critical for pathogenic T helper 17 (Th17) cell migration to the central nervous system (CNS). Whereas many cytokines and their receptors are potently regulated via post-transcriptional mechanisms in response to various stimuli, how CCR6 expression is post-transcriptionally regulated in Th17 cells is unknown. Here, using RNA-binding protein HuR conditional knock-out (KO) and wild-type (WT) mice, we present evidence that HuR post-transcriptionally regulates CCR6 expression by binding to and stabilizing Ccr6 mRNA and by promoting CCR6 translation. We also found that HuR down-regulates several microRNA expressions, which could target the 3'-UTR of Ccr6 mRNA for decay. Accordingly, knock-out of HuR reduced CCR6 expression on Th17 cells and impaired their migration to CNS compared with the response of WT Th17 cells and thereby ameliorated EAE. Together, these findings highlight how HuR contributes to Th17 cell-mediated autoimmune neuroinflammation and support the notion that targeting HuR might be a potential therapeutic intervention for managing autoimmune disorders of the CNS.

Crossing kingdoms: Using decellularized plants as perfusable tissue engineering scaffolds.

Despite significant advances in the fabrication of bioengineered scaffolds for tissue engineering, delivery of nutrients in complex engineered human tissues remains a challenge. By taking advantage of the similarities in the vascular structure of plant and animal tissues, we developed decellularized plant tissue as a prevascularized scaffold for tissue engineering applications. Perfusion-based decellularization was modified for different plant species, providing different geometries of scaffolding. After decellularization, plant scaffolds remained patent and able to transport microparticles. Plant scaffolds were recellularized with human endothelial cells that colonized the inner surfaces of plant vasculature. Human mesenchymal stem cells and human pluripotent stem cell derived cardiomyocytes adhered to the outer surfaces of plant scaffolds. Cardiomyocytes demonstrated contractile function and calcium handling capabilities over the course of 21 days. These data demonstrate the potential of decellularized plants as scaffolds for tissue engineering, which could ultimately provide a cost-efficient, "green" technology for regenerating large volume vascularized tissue mass.

A new glucocerebrosidase-deficient neuronal cell model provides a tool to probe pathophysiology and therapeutics for Gaucher disease.

Glucocerebrosidase is a lysosomal hydrolase involved in the breakdown of glucosylceramide. Gaucher disease, a recessive lysosomal storage disorder, is caused by mutations in the gene GBA1 Dysfunctional glucocerebrosidase leads to accumulation of glucosylceramide and glycosylsphingosine in various cell types and organs. Mutations in GBA1 are also a common genetic risk factor for Parkinson disease and related synucleinopathies. In recent years, research on the pathophysiology of Gaucher disease, the molecular link between Gaucher and Parkinson disease, and novel therapeutics, have accelerated the need for relevant cell models with GBA1 mutations. Although induced pluripotent stem cells, primary rodent neurons, and transfected neuroblastoma cell lines have been used to study the effect of glucocerebrosidase deficiency on neuronal function, these models have limitations because of challenges in culturing and propagating the cells, low yield, and the introduction of exogenous mutant GBA1 To address some of these difficulties, we established a high yield, easy-to-culture mouse neuronal cell model with nearly complete glucocerebrosidase deficiency representative of Gaucher disease. We successfully immortalized cortical neurons from embryonic null allele gba(-/-) mice and the control littermate (gba(+/+)) by infecting differentiated primary cortical neurons in culture with an EF1α-SV40T lentivirus. Immortalized gba(-/-) neurons lack glucocerebrosidase protein and enzyme activity, and exhibit a dramatic increase in glucosylceramide and glucosylsphingosine accumulation, enlarged lysosomes, and an impaired ATP-dependent calcium-influx response; these phenotypical characteristics were absent in gba(+/+) neurons. This null allele gba(-/-) mouse neuronal model provides a much-needed tool to study the pathophysiology of Gaucher disease and to evaluate new therapies.

High-throughput imaging method for direct assessment of GM1 ganglioside levels in mammalian cells.

GM1-gangliosidosis is an inherited autosomal recessive disorder caused by mutations in the gene GLB1, which encodes acid ß-galactosidase (ß-gal). The lack of activity in this lysosomal enzyme leads to accumulation of GM1 gangliosides (GM1) in cells. We have developed a high-content-imaging method to assess GM1 levels in fibroblasts that can be used to evaluate substrate reduction in treated GLB1(-/-) cells [1]. This assay allows fluorescent quantification in a multi-well system which generates unbiased and statistically significant data. Fluorescently labeled Cholera Toxin B subunit (CTXB), which specifically binds to GM1 gangliosides, was used to detect in situ GM1 levels in a fixed monolayer of fibroblasts. This sensitive, rapid, and inexpensive method facilitates in vitro drug screening in a format that allows a high number of replicates using low working volumes.

Enzyme replacement for GM1-gangliosidosis: Uptake, lysosomal activation, and cellular disease correction using a novel ß-galactosidase:RTB lectin fusion.

New enzyme delivery technologies are required for treatment of lysosomal storage disorders with significant pathologies associated with the so-called "hard-to-treat" tissues and organs. Genetic deficiencies in the GLB1 gene encoding acid ß-galactosidase lead to GM1-gangliosidosis or Morquio B, lysosomal diseases with predominant disease manifestation associated with the central nervous system or skeletal system, respectively. Current lysosomal ERTs are delivered into cells based on receptor-mediated endocytosis and do not effectively address several hard-to-treat organs including those critical for GM1-gangliosidosis patients. Lectins provide alternative cell-uptake mechanisms based on adsorptive-mediated endocytosis and thus may provide unique biodistribution for lysosomal disease therapeutics. In the current study, genetic fusions of the plant galactose/galactosamine-binding lectin, RTB, and the human acid ß-galactosidase enzyme were produced using a plant-based bioproduction platform. ß-gal:RTB and RTB:ß-gal fusion products retained both lectin activity and ß-galactosidase activity. Purified proteins representing both fusion orientations were efficiently taken up into GM1 patient fibroblasts and mediated the reduction of GM1 ganglioside substrate with activities matching mammalian cell-derived ß-galactosidase. In contrast, plant-derived ß-gal alone was enzymatically active but did not mediate uptake or correction indicating the need for either lectin-based (plant product) or mannose-6-phosphate-based (mammalian product) delivery. Native ß-galactosidase undergoes catalytic activation (cleavage within the C-terminal region) in lysosomes and is stabilized by association with protective protein/cathepsin A. Enzymatic activity and lysosomal protein processing of the RTB fusions were assessed following internalization into GM1 fibroblasts. Within 1-4h, both ß-gal:RTB and RTB:ß-gal were processed to the ~64kDa "activated" ß-gal form; the RTB lectin was cleaved and rapidly degraded. The activated ß-gal was still detected at 48h suggesting interactions with protective protein/cathepsin A. Uptake-saturation analyses indicated that the RTB adsorptive-mediated mechanisms of ß-gal:RTB supported significantly greater accumulation of ß-galactose activity in fibroblasts compared to the receptor-mediated mechanisms of the mammalian cell-derived ß-gal. These data demonstrate that plant-made ß-gal:RTB functions as an effective replacement enzyme for GM1-gangliosidosis - delivering enzyme into cells, enabling essential lysosomal processing, and mediating disease substrate clearance at the cellular level. RTB provides novel uptake behaviors and thus may provide new receptor-independent strategies that could broadly impact lysosomal disease treatments.

RTB Lectin: a novel receptor-independent delivery system for lysosomal enzyme replacement therapies.

Enzyme replacement therapies have revolutionized patient treatment for multiple rare lysosomal storage diseases but show limited effectiveness for addressing pathologies in "hard-to-treat" organs and tissues including brain and bone. Here we investigate the plant lectin RTB as a novel carrier for human lysosomal enzymes. RTB enters mammalian cells by multiple mechanisms including both adsorptive-mediated and receptor-mediated endocytosis, and thus provides access to a broader array of organs and cells. Fusion proteins comprised of RTB and human α-L-iduronidase, the corrective enzyme for Mucopolysaccharidosis type I, were produced using a tobacco-based expression system. Fusion products retained both lectin selectivity and enzyme activity, were efficiently endocytosed into human fibroblasts, and corrected the disease phenotype of mucopolysaccharidosis patient fibroblasts in vitro. RTB-mediated delivery was independent of high-mannose and mannose-6-phosphate receptors, which are exploited for delivery of currently approved lysosomal enzyme therapeutics. Thus, the RTB carrier may support distinct in vivo pharmacodynamics with potential to address hard-to-treat tissues.

SPARCoC: a new framework for molecular pattern discovery and cancer gene identification.

It is challenging to cluster cancer patients of a certain histopathological type into molecular subtypes of clinical importance and identify gene signatures directly relevant to the subtypes. Current clustering approaches have inherent limitations, which prevent them from gauging the subtle heterogeneity of the molecular subtypes. In this paper we present a new framework: SPARCoC (Sparse-CoClust), which is based on a novel Common-background and Sparse-foreground Decomposition (CSD) model and the Maximum Block Improvement (MBI) co-clustering technique. SPARCoC has clear advantages compared with widely-used alternative approaches: hierarchical clustering (Hclust) and nonnegative matrix factorization (NMF). We apply SPARCoC to the study of lung adenocarcinoma (ADCA), an extremely heterogeneous histological type, and a significant challenge for molecular subtyping. For testing and verification, we use high quality gene expression profiling data of lung ADCA patients, and identify prognostic gene signatures which could cluster patients into subgroups that are significantly different in their overall survival (with p-values < 0.05). Our results are only based on gene expression profiling data analysis, without incorporating any other feature selection or clinical information; we are able to replicate our findings with completely independent datasets. SPARCoC is broadly applicable to large-scale genomic data to empower pattern discovery and cancer gene identification.

Bridger: a new framework for de novo transcriptome assembly using RNA-seq data.

We present a new de novo transcriptome assembler, Bridger, which takes advantage of techniques employed in Cufflinks to overcome limitations of the existing de novo assemblers. When tested on dog, human, and mouse RNA-seq data, Bridger assembled more full-length reference transcripts while reporting considerably fewer candidate transcripts, hence greatly reducing false positive transcripts in comparison with the state-of-the-art assemblers. It runs substantially faster and requires much less memory space than most assemblers. More interestingly, Bridger reaches a comparable level of sensitivity and accuracy with Cufflinks. Bridger is available at https://sourceforge.net/projects/rnaseqassembly/files/?source=navbar.

Big data - a 21st century science Maginot Line? No-boundary thinking: shifting from the big data paradigm.

Whether your interests lie in scientific arenas, the corporate world, or in government, you have certainly heard the praises of big data: Big data will give you new insights, allow you to become more efficient, and/or will solve your problems. While big data has had some outstanding successes, many are now beginning to see that it is not the Silver Bullet that it has been touted to be. Here our main concern is the overall impact of big data; the current manifestation of big data is constructing a Maginot Line in science in the 21st century. Big data is not "lots of data" as a phenomena anymore; The big data paradigm is putting the spirit of the Maginot Line into lots of data. Big data overall is disconnecting researchers and science challenges. We propose No-Boundary Thinking (NBT), applying no-boundary thinking in problem defining to address science challenges.

No-boundary thinking in bioinformatics research.

Currently there are definitions from many agencies and research societies defining "bioinformatics" as deriving knowledge from computational analysis of large volumes of biological and biomedical data. Should this be the bioinformatics research focus? We will discuss this issue in this review article. We would like to promote the idea of supporting human-infrastructure (HI) with no-boundary thinking (NT) in bioinformatics (HINT).

Tools of the trade: developing antibody-based detection capabilities for recombinant proteins.

Protein-specific antibodies serve as critical tools for detection, quantification, and characterization of recombinant proteins. Perhaps the most important and widely used antibody-based procedures for recombinant protein applications are Western immunoblotting and enzyme-linked immunosorbent assays (ELISAs). These analyses require well-characterized, sensitive, and high-affinity antibodies that specifically and selectively recognize the recombinant target protein in the native or denatured form. Although the number of commercially available antibodies is quite substantial and rapidly growing, the appropriate antibody tools for many applications currently do not exist. In this chapter, strategies to develop and characterize both polyclonal and monoclonal antibodies directed against a specific protein of interest are discussed. Experimental strategies and methods are presented for producing and selecting the best antibodies and optimizing protocols for Western analyses, ELISAs, and other applications. Once antibody and procedure optimization is completed to ensure specificity, sensitivity, accuracy, and reliability, these immune-based approaches can now serve as powerful and enabling tools in the characterization, detection and diagnostics, structure/function analysis, and quality assessment of recombinant proteins.

Quality assessment of recombinant proteins produced in plants.

Plant-based expression technologies for recombinant proteins have begun to receive acceptance for pharmaceuticals and other commercial markets. Protein products derived from plants offer safer, more cost-effective, and less capital-intensive alternatives to traditional manufacturing systems using microbial fermentation or animal cell culture bioreactors. Moreover, plants are now known to be capable of expressing bioactive proteins from a diverse array of species including animals and humans. Methods development to assess the quality and performance of proteins manufactured in plants are essential to support the QA/QC demands as plant-produced protein products transition to the commercial marketplace. Within the pharmaceutical arena, process validation and acceptance criteria for biological products must comply with the Food and Drug Administration (FDA) and ICH Q6B guidelines in order to initiate the regulatory approval process. Detailed product specifications will also need to be developed and validated for plant-made proteins for the bioenergy, food, chemical synthesis, or research reagent markets.We have, therefore, developed assessment methods for important qualitative and quantitative parameters of the products and the manufacturing methods utilized in plant-based production systems. In this chapter, we describe a number of procedures to validate product identity and characteristics including mass analyses, antibody cross-reactivity, N-terminal sequencing, and bioactivity. We also address methods for routine assessment of yield, recovery, and purity. The methods presented are those developed for the synthesis and recovery of the avian cytokine, chicken interleukin-12 (ChIL-12), produced in the leaves of Nicotiana benthamiana. The ChIL-12 protein used as a model for this chapter includes a C-terminal histidine epitope (HIS-tag) and, thus, these methods may be directly applicable to other HIS-tagged proteins produced in plants. However, the overall strategy presented using the ChIL-12(HIS) example should provide the basis of standard procedures for assessing the quality of other plant-based protein products and manufacturing systems.

Green factory: plants as bioproduction platforms for recombinant proteins.

Molecular farming, long considered a promising strategy to produce valuable recombinant proteins not only for human and veterinary medicine, but also for agriculture and industry, now has some commercially available products. Various plant-based production platforms including whole-plants, aquatic plants, plant cell suspensions, and plant tissues (hairy roots) have been compared in terms of their advantages and limits. Effective recombinant strategies are summarized along with descriptions of scalable culture systems and examples of commercial progress and success.

TERPRED: A Dynamic Structural Data Analysis Tool.

Computational protein structure prediction mainly involves the main-chain prediction and the side-chain confirmation determination. In this research, we developed a new structural bioinformatics tool, TERPRED for generating dynamic protein side-chain rotamer libraries. Compared with current various rotamer sampling methods, our work is unique in that it provides a method to generate a rotamer library dynamically based on small sequence fragments of a target protein. The Rotamer Generator provides a means for existing side-chain sampling methods using static pre-existing rotamer libraries, to sample from dynamic target-dependent libraries. Also, existing side-chain packing algorithms that require large rotamer libraries for optimal performance, could possibly utilize smaller, target-relevant libraries for improved speed.

Movement of protein and macromolecules between host plants and the parasitic weed Phelipanche aegyptiaca Pers.

Little is known about the translocation of proteins and other macromolecules from a host plant to the parasitic weed Phelipanche spp. Long-distance movement of proteins between host and parasite was explored using transgenic tomato plants expressing green fluorescent protein (GFP) in their companion cells. We further used fluorescent probes of differing molecular weights to trace vascular continuity between the host plant and the parasite. Accumulation of GFP was observed in the central vascular bundle of leaves and in the root phloem of transgenic tomato plants expressing GFP under the regulation of AtSUC2 promoter. When transgenic tomato plants expressing GFP were parasitized with P. aegyptiaca, extensive GFP was translocated from the host phloem to the parasite phloem and accumulated in both Phelipanche tubercles and shoots. No movement of GFP to the parasite was observed when tobacco plants expressing GFP targeted to the ER were parasitized with P. aegyptiaca. Experiments using fluorescent probes of differing molecular weights to trace vascular continuity between the host plant and the parasite demonstrated that Phelipanche absorbs dextrans up to 70 kDa in size from the host and that this movement can be bi-directional. In the present study, we prove for the first time delivery of proteins from host to the parasitic weed P. aegyptiaca via phloem connections, providing information for developing parasite resistance strategies.

Efficient plant-based production of chicken interleukin-12 yields a strong immunostimulatory cytokine.

Interleukin-12 (IL-12), an important immunomodulator for cell-mediated immunity, shows significant potential as a vaccine adjuvant and anticancer therapeutic in mammals. Therapeutic strategies to develop mammalian IL-12 as a vaccine adjuvant/immunomodulator for promoting cellular immunity and establishing a Th1-biased immune response further support the potential value of ChIL-12. Transgenic plants show promise as scalable bioproduction platforms for challenging biopharmaceutical proteins. We have expressed, characterized, and purified biologically active ChIL-12 in plants using a rapid Agrobacterium-mediated tobacco plant-based transient expression system. To ensure the stoichiometric expression and assembly of p35 and p40, we expressed a single-chain version of chicken IL-12 (ChIL-12). A histidine 6x tag was used for identity and purification of ChIL-12(His) protein. Our results demonstrated precise cleavage of the endogenous chicken p40 signal peptide in plants as well as addition of N-linked glycans. Biological activity was confirmed in vitro by interferon-gamma secretion of ChIL-12-treated chicken splenocytes. In addition, splenocytes treated with ChIL-12 expressed with or without the His tag demonstrated comparable ChIFN-gamma induction. These studies indicate that plant-based platforms for bioproduction of complex pharmaceutical proteins produce functional ChIL-12 and provide key advantages in safety, scale, and cost-effective platform for veterinary vaccine and therapeutic applications.

Rapid system for evaluating bioproduction capacity of complex pharmaceutical proteins in plants.

Transgene product yield remains a key limitation in commercializing plant-derived pharmaceutical proteins. Although significant progress has been made in understanding the roles of promoters, enhancers, integration sites, codon usage, cryptic RNA sites, silencing, and product compartmentalization on product yield and quality, researchers still cannot reliably predict which proteins will be produced at high levels or what manipulations will guarantee enhanced productivity. We have optimized a simple transient expression system in Nicotiana benthamiana enabling rapid assessment of transgene potential for plant-based bioproduction. Briefly, intact Nicotiana benthamiana plants are vacuum-infiltrated with Agrobacterium tumefaciens cultures carrying the transgene of interest. After 48-96 h of further incubation, leaves are harvested for protein characterization. Using the immunomodulator interleukin-12 as a model pharmaceutical protein, we obtained bioactive recombinant protein at levels exceeding 5% of total soluble leaf protein. Appropriately assembled multimeric proteins have also been obtained following coinfiltration with Agrobacterium tumefaciens strains individually encoding each subunit. This system provides a rapid source of transgene product for assessing posttranslational modifications, purification strategies, and bioactivity as well as an effective system for optimizing construct elements. For vaccines, product purified from two to eight plants may support mouse vaccination trials providing efficacy and immune assessment data early in the development process.

Functional characterization of a stilbene synthase gene using a transient expression system in planta.

The expression and functionality of a resveratrol synthase (RS) gene from peanut (Arachis hypogaea) was studied using an Agrobacterium tumefaciens-mediated transient expression system in Nicotiana benthamiana leaves. Functional analysis of RS was demonstrated by tracking its expression during 96 h. To measure the transcripts levels of RS transgene, real-time qRT-PCR was used and revealed that the highest level of transcripts was at 48 h post-transfection. Western blot analyses showed that RS protein was accumulated to the highest levels at 72 h post-transfection. Finally, HPLC and mass spectrometry analyses revealed the production of trans-piceid (resveratrol glucoside) as the major stilbenoid compound confirming the functional activity of the RS enzyme in planta. No activity of RS transgene was detected in negative controls. This strategy showed advantages over conventional systems because it does not require establishment of cell cultures, feeding with appropriate substrates or generation of stable transgenic plants. This transient system proved to be a rapid and direct approach to perform functional analysis of stilbene synthases, such as resveratrol synthase. Furthermore, this approach can be useful to study the metabolic effects of over-expressing or silencing specific genes within a short period of time.