Expanding the understanding of viral tropism by characterizing the N-glycomic profiles of transducible cell lines and tissue types and Characterizing functional receptors that mediate the inhibitory relationship between 4-O-sulfated chondroitin sulfate and neurons

1: Glycosylation plays an important role in facilitating viral transduction by acting as preliminary cell surface receptors. For this reason, the structural determinants in glycans that dictate viral tissue tropism need to be extensively studied to improve the efficacy of gene therapy vectors in basic research and eventually the clinic. Elucidating the dependencies for viral transduction initiation and understanding how these structural nuances of glycans initiate virion specific tropic effects is paramount when considering how to use vectors to improve clinical outcomes for patients suffering from illnesses with few treatment options. The goal of this project was to use MALDI-TOF-MS to provide baseline N-glycan profiles of the cell lines and tissues used to test gene therapy vectors. In doing so these profiles will be valuable to the field by clarifying what structural determinants may influence viral tropism. It was discovered Neu5Ac sialic acid content differs qualitatively amongst the seven cell lines analyzed. These differences may play into why some cell lines such as CHO-K1 and COS-7 can transduce more preferentially with some AAV serotypes like AAV5. In addition, sialic acid differences were also assessed in three tissue types used in transduction assays. Abstract 2: After injury to the CNS, reactive astrocytes form a protective extracellular matrix to isolate damaged tissue. These astrocytes influence the surrounding tissue by upregulating the production of proteoglycans containing chondroitin sulfate. Due to the new cellular environment, chondroitin sulfate (CS) glycosaminoglycan chains are upregulated with predominately 4-O-sulated sulfation patterns. These sulfation patterns are known to inhibit axonal guidance, and ultimately neuronal regeneration. While the inhibitory effect of CS is well known, the mechanism by which these specific sulfation patterns may interact with receptors also known to have inhibitory effects on neuro-regeneration such as protein tyrosine phosphatase σ is unknown. To characterize these interactions reductive amination was used to immobilize these CS chains onto solid beads. Chondroitin sulfate was isolated from the organs of an ARSB null mouse model which lacks the N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ARSB) which is involved in the degradation of glycosaminoglycans (GAGs). Disruption of arylsulfatase B leads to the production of CS chains with 4-O-sulfated non-reducing ends exclusively. Key findings indicate that purified GAG chains retain their ligand specificity after being covalently immobilized onto solid supports, and that these systems can be utilized to characterize the relationship between inhibitory forms of CS and protein tyrosine phosphatase σ.

Alternative strategy to induce CRISPR-mediated genetic changes in hematopoietic cells

Acute Myeloid Leukaemia is a complex heterogenous disease caused by clonal expansion of undifferentiated myeloid precursors. Recently, several haematological models have been developed with CRISPR/Cas9, using viral vectors, because blood cells are hard to transfect. To avoid virus disadvantages, we have developed a strategy to generate CRISPR constructs, by means of PCR, which any lab equipped with basic technology can implement. These PCR-generated constructs enter easily into hard-to-transfect cells. After testing its functionality by editing MYBL2 gene in HEK293 cells, we successfully introduced the R172 mutation in IDH2 gene in NB4 cells that expresses constitutively the Cas9 nuclease. Comparing our methodology with ribonucleoprotein strategies, we found that mutation introduction efficiency was similar between both methodologies, and no off-target events were detected. Our strategy represents a valid alternative to introduce desired mutations in hard to transfect leukemic cells, avoiding using huge vectors or viral transduction.

Vectofusin-1 based T-cell transduction approach for developing murine CAR-T cells for cancer.

Gene transfer into human and murine T-cells using viral-based approaches has several promising therapeutic applications including the production of chimeric antigen receptor T-cell (CAR-T) therapy. The generation of murine CAR-T is paramount to test and validate immunocompetent mouse models for CAR-T therapy. Several viral transduction enhancers already exist for gene therapy with few limitations. In this study, we tested vectofusin-1, a short cationic peptide, as a soluble transduction enhancer for gammaretroviral transduction for the generation of anti-CD19 murine CAR-T. We found that in comparison to Retronectin, Vectofusin-1 is an equally optimal transduction enhancer for the generation of murine CAR-T cells.

Viral transduction and the dynamics of bacterial adaptation

Viral infections can exert a large influence on their hosts' ecology by causing widespread mortality, but they also shape the evolutionary adaptation of hosts in a number of ways. A major pathway for viruses to do so is through the transfer of genetic material among individual hosts, a process known as transduction. While horizontal gene transfer is known as a major factor in prokaryotic macroevolution, its role in the microevolutionary adaptation of hosts populations is poorly known. By facilitating the transfer of beneficial alleles between host cells, transduction might facilitate and accelerate bacterial adaptation. Conversely, the risk of transferring deleterious alleles may hinder and slow it down. Here we resolve the effect of transduction on bacterial adaptation in a simple eco-evolutionary model for the combined dynamics of transduction and adaptive evolution of an ecological (resource-use) trait. The transfer of beneficial alleles by tranduction speeds up adaptation whereas the transfer of deleterious alleles causes strong stochastic fluctuations of the trait value around the adapted value. In contrast to the expected effect of recombination, which tends to oppose phenotypic diversification, viral transduction can increase host phenotypic diversity.

Immunoproteasome Activity and Content Determine Hematopoietic Cell Sensitivity to ONX-0914 and to the Infection of Cells with Lentiviruses

Proteasomes are intracellular structures responsible for protein degradation. The 20S proteasome is a core catalytic element of the proteasome assembly. Variations of catalytic subunits generate different forms of 20S proteasomes including immunoproteasomes (iPs), which are present mostly in the immune cells. Certain cells of the immune system are primary targets of retroviruses. It has been shown that several viral proteins directly affect proteasome functionality, while inhibition of proteasome activity with broad specificity proteasome inhibitors stimulates viral transduction. Here we specifically addressed the role of the immunoproteasomes during early stages of viral transduction and investigated the effects of specific immunoproteasome inhibition and activation prior to infection using a panel of cell lines. Inhibition of iPs in hematopoietic cells with immunoproteasome-specific inhibitor ONX-0914 resulted in increased infection by VSV-G pseudotyped lentiviruses. Moreover, a tendency for increased infection of cloned cells with endogenously decreased proteasome activity was revealed. Conversely, activation of iPs by IFN-γ markedly reduced the viral infectivity, which was rescued upon simultaneous immunoproteasome inhibition. Our results indicate that immunoproteasome activity might be determinative for the cellular antiretroviral resistance at least for the cells with high iP content. Finally, therapeutic application of immunoproteasome inhibitors might promote retroviral infection of cells in vivo.

Cell-permeable transgelin-2 as a potent therapeutic for dendritic cell-based cancer immunotherapy

Background Transgelin-2 is a 22 kDa actin-binding protein that has been proposed to act as an oncogenic factor, capable of contributing to tumorigenesis in a wide range of human malignancies. However, little is known whether this tiny protein also plays an important role in immunity, thereby keeping body from the cancer development and metastasis. Here, we investigated the functions of transgelin-2 in dendritic cell (DC) immunity. Further, we investigated whether the non-viral transduction of cell-permeable transgelin-2 peptide potentially enhance DC-based cancer immunotherapy. Methods To understand the functions of transgelin-2 in DCs, we utilized bone marrow-derived DCs (BMDCs) purified from transgelin-2 knockout (Tagln2−/−) mice. To observe the dynamic cellular mechanism of transgelin-2, we utilized confocal microscopy and flow cytometry. To monitor DC migration and cognate T–DC interaction in vivo, we used intravital two-photon microscopy. For the solid and metastasis tumor models, OVA+ B16F10 melanoma were inoculated into the C57BL/6 mice via intravenously (i.v.) and subcutaneously (s.c.), respectively. OTI TCR T cells were used for the adoptive transfer experiments. Cell-permeable, de-ubiquitinated recombinant transgelin-2 was purified from Escherichia coli and applied for DC-based adoptive immunotherapy. Results We found that transgelin-2 is remarkably expressed in BMDCs during maturation and lipopolysaccharide activation, suggesting that this protein plays a role in DC-based immunity. Although Tagln2−/− BMDCs exhibited no changes in maturation, they showed significant defects in their abilities to home to draining lymph nodes (LNs) and prime T cells to produce antigen-specific T cell clones, and these changes were associated with a failure to suppress tumor growth and metastasis of OVA+ B16F10 melanoma cells in mice. Tagln2−/− BMDCs had defects in filopodia-like membrane protrusion and podosome formation due to the attenuation of the signals that modulate actin remodeling in vitro and formed short, unstable contacts with cognate CD4+ T cells in vivo. Strikingly, non-viral transduction of cell-permeable, de-ubiquitinated recombinant transgelin-2 potentiated DC functions to suppress tumor growth and metastasis. Conclusion This work demonstrates that transgelin-2 is an essential protein for both cancer and immunity. Therefore, transgelin-2 can act as a double-edged sword depending on how we apply this protein to cancer therapy. Engineering and clinical application of this protein may unveil a new era in DC-based cancer immunotherapy. Our findings indicate that cell-permeable transgelin-2 have a potential clinical value as a cancer immunotherapy based on DCs.

Syntenin-knock out reduces exosome turnover and viral transduction

Exosomal transfers represent an important mode of intercellular communication. Syntenin is a small scaffold protein that, when binding ALIX, can direct endocytosed syndecans and syndecan cargo to budding endosomal membranes, supporting the formation of intraluminal vesicles that compose the source of a major class of exosomes. Syntenin, however, can also support the recycling of these same components to the cell surface. Here, by studying mice and cells with syntenin-knock out, we identify syntenin as part of dedicated machinery that integrates both the production and the uptake of secreted vesicles, supporting viral/exosomal exchanges. This study significantly extends the emerging role of heparan sulfate proteoglycans and syntenin as key components for macromolecular cargo internalization into cells.

Generation of stably expressing IRF5 spliced isoform in Jurkat cells

Lentiviral transduction enables the generation of gain-of-function of a targeted gene in mammalian cells. Single-cell cloning through limiting dilution can establish a population of cells with homogenous transgene expression for exploring protein function. Here, we describe step by step optimized protocols for generating clonal stably expressing using crude lentiviral supernatant in Jurkat cells. Although the protocol is for general use, we will detail how to create stable cell lines based on Jurkat cells expressing IRF5 spliced isoform. These protocols will be broadly useful for researchers seeking to apply overexpression by viral transduction and generation of stable clone to study gene function in mammalian cells.