Gene transfer has therapeutic potential for treating HIV-1 infection by generating cells that are resistant to the virus. We have engineered a novel self-inactivating lentiviral vector, LVsh5/C46, using two viral-entry inhibitors to block early steps of HIV-1 cycle. The LVsh5/C46 vector encodes a short hairpin RNA (shRNA) for downregulation of CCR5, in combination with the HIV-1 fusion inhibitor, C46. We demonstrate here the effective delivery of LVsh5/C46 to human T cell lines, peripheral blood mononuclear cells, primary CD4+ T lymphocytes, and CD34+ hematopoietic stem/progenitor cells (HSPC). CCR5-targeted shRNA (sh5) and C46 peptide were stably expressed in the target cells and were able to effectively protect gene-modified cells against infection with CCR5- and CXCR4-tropic strains of HIV-1. LVsh5/C46 treatment was nontoxic as assessed by cell growth and viability, was noninflammatory, and had no adverse effect on HSPC differentiation. LVsh5/C46 could be produced at a scale sufficient for clinical development and resulted in active viral particles with very low mutagenic potential and the absence of replication-competent lentivirus. Based on these in vitro results, plus additional in vivo safety and efficacy data, LVsh5/C46 is now being tested in a phase 1/2 clinical trial for the treatment of HIV-1 disease.

These findings validate the potential of this methodology for developing a gene transfer treatment for CF airway disease.

OBJECTIVE MicroRNAs (miRNAs) regulate gene expression and therefore play important roles in many physiological and pathological processes. The aim of this pilot study was to determine the feasibility of extraction and subsequent profiling of miRNA from CSF samples in a pilot population of aneurysmal subarachnoid hemorrhage patients and establish if there is a distinct CSF miRNA signature between patients who develop cerebral vasospasm and those who do not. METHODS CSF samples were taken at various time points during the clinical management of a subset of SAH patients (SAH patient samples without vasospasm, n = 10; SAH patient samples with vasospasm, n = 10). CSF obtained from 4 patients without SAH was also included in the analysis. The miRNA was subsequently isolated and purified and then analyzed on an nCounter instrument using the Human V2 and V3 miRNA assay kits. The data were imported into the nSolver software package for differential miRNA expression analysis. RESULTS From a total of 800 miRNAs that could be detected with each version of the miRNA assay kit, a total of 691 miRNAs were communal to both kits. There were 36 individual miRNAs that were differentially expressed (p < 0.01) based on group analyses, with a number of miRNAs showing significant changes in more than one group analysis. The changes largely reflected differences between non-SAH and SAH groups. These included miR-204-5p, miR-223-3p, miR-337-5p, miR-451a, miR-489, miR-508-3p, miR-514-3p, miR-516-5p, miR-548 m, miR-599, miR-937, miR-1224-3p, and miR-1301. However, a number of miRNAs did exclusively differ between the vasospasm and nonvasospasm SAH groups including miR-27a-3p, miR-516a-5p, miR-566, and miR-1197. CONCLUSIONS The findings indicate that temporal miRNA profiling can detect differences between CSF from aneurysmal SAH and non-SAH patients. Moreover, the miRNA profile of CSF samples from patients who develop cerebral vasopasm may be distinguishable from those who do not. These results provide a foundation for future research at identifying novel CSF biomarkers that might predispose to the development of cerebral vasospasm after SAH and therefore influence subsequent clinical management.

By taking a systematic approach to optimising all aspects of our five-plasmid lentiviral vector system we improved titre, safety, large-scale production, and demonstrated that infectivity could be specifically controlled.

Natural killer (NKs) cells provide rapid responses to viral-infected and malignant cells, including acute myeloid leukemia (AML) blasts. The balance among inhibitory and activating signals, delivered by multiple interactions between ligands on target cells and NK receptors, determines the posture of the NK cell response to either one of target cell elimination or tolerance. The aim of this work was to study the influence of the differential expression of activating and inhibitory NK receptor ligands (NKRLs) by leukemic blasts on clinical outcome in newly diagnosed AML patients. Leukemic cells and clinical data from 66 patients undergoing induction chemotherapy were obtained from the Australasian Leukemia Lymphoma Group tissue bank. Expression of 6 activating (MICA, MICAB, CD155, CD112, ULBP1, and ULBP2/5/6) and 3 inhibitory (HLA class I, PD-L1, and PD-L2) NKRLs was analyzed by flow cytometry. AML blasts displayed heterogeneous expression of NKRLs. MICA, CD112, and ULBP1 were most frequently expressed. ULBP1 expression was significantly associated with improved 2-year overall survival (51.4% vs 11.4%), relapse-free survival (42.5% vs 10.0%), and reduced relapse (44.1% vs 78.6%). We calculated a net score of activating minus inhibitory ligands and demonstrated that the expression of an overall activating NK ligand phenotype was associated with superior 2-year overall survival (59.6% vs 24.4%) and reduced relapse (31.5% vs 68.2%). Our study provides clinical evidence for the role of NK cell-mediated immunoediting against AML, mediated by the expression of NKRLs on blasts, and supports investigation into strategies to enhance NK cell function to improve outcomes in patients with AML.

Glioblastoma is the most aggressive and lethal tumour of the central nervous system and as such the identification of reliable prognostic and predictive biomarkers for patient survival and tumour recurrence is paramount. MicroRNA detection has rapidly emerged as potential biomarkers, in patients with glioblastoma. Over the last decade, analysis of miRNA in laboratory based studies have yielded several candidates as potential biomarkers however, the accepted use of these candidates in the clinic is yet to be validated. Here we will examine the use of miRNA signatures to improve glioblastoma stratification into subgroups and summarise recent advances made in miRNA examination as potential biomarkers for glioblastoma progression and recurrence.

Constantine S. Tam, and Ilia Voskoboinik. Differential effects of BTK inhibitors ibrutinib and zanubrutinib on NK cell effector function in patients with mantle cell lymphoma.

Gene therapy of the cornea shows promise for modulating corneal transplant rejection but the most appropriate vector for gene transfer has yet to be determined. We investigated a lentiviral vector (LV) for its ability to transduce corneal endothelium. A lentivector expressing enhanced yellow fluorescent protein (eYFP) under the control of the Simian virus type 40 early promoter (LV-SV40-eYFP) transduced 80-90% of rat, ovine and human corneal endothelial cells as detected by fluorescence microscopy. The kinetics of gene expression varied among species, with ovine corneal endothelium showing a relative delay in detectable reporter gene expression compared with the rat or human corneal endothelium. Vectors containing the myeloproliferative sarcoma virus promoter or the phosphoglycerate kinase promoter were not significantly more effective than LV-SV40-eYFP. The stability of eYFP expression in rat and ovine corneas following ex vivo transduction of the donor cornea was assessed following orthotopic corneal transplantation. Following transduction ex vivo, eYFP expression was maintained in corneal endothelial cells for at least 28 days after corneal transplantation in the sheep and 460 days in the rat. Thus, rat, ovine and human corneal endothelial cells were efficiently transduced by the LV, and gene expression appeared stable over weeks in vivo. Gene Therapy (2007) 14, 760-767.