Preclinical Biodistribution and Safety Evaluation of a pbi-shRNA STMN1 Lipoplex after Subcutaneous Delivery.

Stathmin-1 (STMN1) is a microtubule-destabilizing protein which is overexpressed in cancer. Its overexpression is associated with poor prognosis and also serves as a predictive marker to taxane therapy. We have developed a proprietary bi-functional shRNA (bi-shRNA) platform to execute RNA interference (RNAi)-mediated gene silencing and a liposome-carrier complex to systemically deliver the pbi-shRNA plasmids.

Phase II study of Vigil® DNA engineered immunotherapy as maintenance in advanced stage ovarian cancer.

Objectives: The majority of women with Stage III/IV ovarian cancer who achieve clinical complete response with frontline standard of care will relapse within 2years. Vigil immunotherapy, a GMCSF/bi-shRNA furin DNA engineered autologous tumor cell (EATC) product, demonstrated safety and induction of circulating activated T-cells against autologous tumor in Phase I trial Senzer et al. (2012, 2013) .

Preclinical Justification of pbi-shRNA EWS/FLI1 Lipoplex (LPX) Treatment for Ewing's Sarcoma.

The EWS/FLI1 fusion gene is well characterized as a driver of Ewing's sarcoma. Bi-shRNA EWS/FLI1 is a functional plasmid DNA construct that transcribes both siRNA and miRNA-like effectors each of which targets the identical type 1 translocation junction region of the EWS/FLI1 transcribed mRNA sequence. Previous preclinical and clinical studies confirm the safety of this RNA interference platform technology and consistently demonstrate designated mRNA and protein target knockdown at greater than 90% efficiency.

Pilot Trial of FANG Immunotherapy in Ewing's Sarcoma.

We report on 12 consecutive patients with advanced/metastatic Ewing's sarcoma who were treated as a separate cohort of a phase 1 trial of FANG autologous immunotherapy (1 × 10(6)-2.5 × 10(7) cells/intradermal injection each month for minimum 4 months). Safety and clinical response were monitored.

Phase 1 Trial of Bi-shRNA STMN1 BIV in Refractory Cancer.

Stathmin1 (STMN1) is a microtubule modulator that is expressed in multiple cancers and correlates with poor survival. We previously demonstrated in vivo safety of bifunctional (bi) shRNA STMN1 bilamellar invaginated vesicle (BIV) and that systemic delivery correlated with antitumor activity. Patients with superficial advanced refractory cancer with no other standard options were entered into trial.

Summary of bi-shRNA/GM-CSF augmented autologous tumor cell immunotherapy (FANG™) in advanced cancer of the liver.

Therapies for advanced hepatocellular carcinoma (HCC) are limited. We carried out a phase I trial of a novel autologous whole-cell tumor cell immunotherapy (FANG™), which incorporates a dual granulocyte macrophage colony-stimulating factor (GM-CSF) expressive/bifunctional small hairpin RNA interference (bi-shRNAi) vector. The bi-shRNAi DNA targets furin, which is a proconvertase of transforming growth factors beta (TGFβ) 1 and 2.

Bifunctional short hairpin RNA (bi-shRNA): design and pathway to clinical application.

The discovery of RNA interference (RNAi) engendered great excitement and raised expectations regarding its potential applications in biomedical research and clinical usage. Over the ensuing years, expanded understanding of RNAi and preliminary results from early clinical trials tempered enthusiasm with realistic appraisal resulting in cautious optimism and a better understanding of necessary research and clinical directions. As a result, data from more recent trials are beginning to show encouraging positive clinical outcomes.

Phase I trial of "bi-shRNAi(furin)/GMCSF DNA/autologous tumor cell" vaccine (FANG) in advanced cancer.

We performed a phase I trial of FANG vaccine, an autologous tumor-based product incorporating a plasmid encoding granulocyte-macrophage colony-stimulating factor (GMCSF) and a novel bifunctional short hairpin RNAi (bi-shRNAi) targeting furin convertase, thereby downregulating endogenous immunosuppressive transforming growth factors (TGF) β1 and β2. Patients with advanced cancer received up to 12 monthly intradermal injections of FANG vaccine (1 × 10(7) or 2.5 × 10(7) cells/ml injection).

In vivo safety and antitumor efficacy of bifunctional small hairpin RNAs specific for the human Stathmin 1 oncoprotein.

Bifunctional small hairpin RNAs (bi-shRNAs) are functional miRNA/siRNA composites that are optimized for posttranscriptional gene silencing through concurrent mRNA cleavage-dependent and -independent mechanisms (Rao et al., 2010 ). We have generated a novel bi-shRNA using the miR30 scaffold that is highly effective for knockdown of human stathmin (STMN1) mRNA.

Phase I trial of TGF-beta 2 antisense GM-CSF gene-modified autologous tumor cell (TAG) vaccine.

Purpose: On the basis of the hypothesis that the combined expression of immunostimulatory granulocyte macrophage colony stimulating factor (GM-CSF) and antitumor suppressor TGF-β2 antisense (AS) transgenes can break tolerance and stimulate immune responses to cancer-associated antigens, we constructed an expression plasmid [the tumor-associated glycoprotein (TAG) plasmid] that coexpresses GM-CSF and TGF-β2 AS nucleotide sequences and which was incorporated into an autologous whole-cell vaccine.

Experimental Design: Patients undergoing resection were enrolled. Freshly harvested autologous tumor cells were mechanically and enzymatically disaggregated, then electroporated with the TAG vector.

Phase II study of belagenpumatucel-L, a transforming growth factor beta-2 antisense gene-modified allogeneic tumor cell vaccine in non-small-cell lung cancer.

Purpose: Belagenpumatucel-L is a nonviral gene-based allogeneic tumor cell vaccine that demonstrates enhancement of tumor antigen recognition as a result of transforming growth factor beta-2 inhibition.

Patients And Methods: We performed a randomized, dose-variable, phase II trial involving stages II, IIIA, IIIB, and IV non-small-cell lung cancer patients. Each patient received one of three doses (1.

Immunoglobulin M myeloma: evaluation of molecular features and cytokine expression.

Immunoglobulin (Ig) M myeloma is a distinct entity with features of multiple myeloma (MM) and Waldenstrom's macroglobulinemia (WM). The malignant cells in IgM myeloma have a distinctive chromosomal translocation that differentiates them from WM. These cells are postgerminal-center in origin with isotype-switch transcripts.

Functional modulation of smooth muscle cells by the inflammatory mediator CAP37.

CAP37, a neutrophil-derived protein, originally identified for its antimicrobial activity is now known to have strong immunoregulatory effects on host cells. Recently, we described its expression and localization within the vascular endothelium associated with atherosclerotic plaques. Since CAP37 is a potent activator of endothelial cells and monocytes, two of the key cellular components of the atherosclerotic plaque, this study was undertaken to determine whether CAP37 had functional effects on smooth muscle cells another important cellular participant in atherosclerosis.

CAP37, a neutrophil-derived inflammatory mediator, augments leukocyte adhesion to endothelial monolayers.

Cationic antimicrobial protein of molecular weight 37 kDa (CAP37) is a multifunctional inflammatory mediator that was originally isolated from human neutrophils and described to possess bactericidal and monocyte-activating functions. More recently its expression in endothelial and epithelial cells in response to inflammatory mediators and its ability to activate endothelial cells and alter permeability has been demonstrated. We hypothesize that CAP37 facilitates the process of transendothelial migration not only because of its potential to act as a chemoattractant but also through its ability to promote leukocyte adhesion to the endothelium by modulating adhesion molecule expression on the endothelium.

Activation of microglia: a neuroinflammatory role for CAP37.

Recent evidence suggests that inflammation and immune function in the central nervous system (CNS) may play a considerable role in the progression of many neurodegenerative diseases. It is known that microglia, the CNS equivalent of peripheral blood monocytes, may be instrumental in causing neurotoxicity. However, the mediator(s) that activates microglia to produce toxic substances that orchestrate cell death has yet to be elucidated.

Corneal expression of the inflammatory mediator CAP37.

Purpose: CAP37 is a polymorphonuclear neutrophil (PMN)-derived inflammatory protein with potent antibiotic and chemotactic activity. To further investigate the biological significance of CAP37 in infection and inflammation, a well-characterized in vivo rabbit model of bacterial keratitis was selected to study its contribution to host defenses.

Methods: One hundred colony-forming units of log phase Staphylococcus aureus was injected intrastromally.

CAP37, a novel inflammatory mediator: its expression in endothelial cells and localization to atherosclerotic lesions.

Cationic antimicrobial protein of 37 kd (CAP37), originally isolated from human neutrophils, is an important multifunctional inflammatory mediator. Here we describe its localization within the vascular endothelium associated with atherosclerotic plaques. Evidence from in vitro immunocytochemical, Northern blot, and reverse transcriptase-polymerase chain reaction analysis indicates that CAP37 is induced in endothelial cells in response to inflammatory mediators.