In-depth proteomic analyses of direct expressed prostatic secretions.

It is expected that clinically obtainable fluids that are proximal to organs contain a repertoire of secreted proteins and shed cells reflective of the physiological state of that tissue and thus represent potential sources for biomarker discovery, investigation of tissue-specific biology, and assay development. The prostate gland secretes many proteins into a prostatic fluid that combines with seminal vesicle fluids to promote sperm activation and function. Proximal fluids of the prostate that can be collected clinically are seminal plasma and expressed prostatic secretion (EPS) fluids. In the current study, MudPIT-based proteomics was applied to EPS obtained from nine men with prostate cancer and resulted in the confident identification of 916 unique proteins. Systematic bioinformatics analyses using publicly available microarray data of 21 human tissues (Human Gene Atlas), the Human Protein Atlas database, and other published proteomics data of shed/secreted proteins were performed to systematically analyze this comprehensive proteome. Therefore, we believe this data will be a valuable resource for the research community to study prostate biology and potentially assist in the identification of novel prostate cancer biomarkers. To further streamline this process, the entire data set was deposited to the Tranche repository for use by other researchers.

Direct Lymph Node Vaccination of Lentivector/Prostate-Specific Antigen is Safe and Generates Tissue-Specific Responses in Rhesus Macaques.

Anti-cancer immunotherapy is emerging from a nadir and demonstrating tangible benefits to patients. A variety of approaches are now employed. We are invoking antigen (Ag)-specific responses through direct injections of recombinant lentivectors (LVs) that encode sequences for tumor-associated antigens into multiple lymph nodes to optimize immune presentation/stimulation. Here we first demonstrate the effectiveness and antigen-specificity of this approach in mice challenged with prostate-specific antigen (PSA)-expressing tumor cells. Next we tested the safety and efficacy of this approach in two cohorts of rhesus macaques as a prelude to a clinical trial application. Our vector encodes the cDNA for rhesus macaque PSA and a rhesus macaque cell surface marker to facilitate vector titering and tracking. We utilized two independent injection schemas demarcated by the timing of LV administration. In both cohorts we observed marked tissue-specific responses as measured by clinical evaluations and magnetic resonance imaging of the prostate gland. Tissue-specific responses were sustained for up to six months-the end-point of the study. Control animals immunized against an irrelevant Ag were unaffected. We did not observe vector spread in test or control animals or perturbations of systemic immune parameters. This approach thus offers an "off-the-shelf" anti-cancer vaccine that could be made at large scale and injected into patients-even on an out-patient basis.

Autologous transplantation of lentivector/acid ceramidase-transduced hematopoietic cells in nonhuman primates.

Farber disease is a rare lysosomal storage disorder (LSD) that manifests due to acid ceramidase (AC) deficiencies and ceramide accumulation. We present a preclinical gene therapy study for Farber disease employing a lentiviral vector (LV-huAC/huCD25) in three enzymatically normal nonhuman primates. Autologous, mobilized peripheral blood (PB) cells were transduced and infused into fully myelo-ablated recipients with tracking for at least 1 year. Outcomes were assessed by measuring the AC specific activity, ceramide levels, vector persistence/integration, and safety parameters. We observed no hematological, biochemical, radiological, or pathological abnormalities. Hematological recovery occurred by approximately 3 weeks. Vector persistence was observed in PB and bone marrow (BM) cells by qualitative and quantitative PCR. We did not observe any clonal proliferation of PB and BM cells. Importantly, AC-specific activity was detected above normal levels in PB and BM cells analyzed post-transplantation and in spleens and livers at the endpoint of the study. Decreases of ceramide in PB cells as well as in spleen and liver tissues were seen. We expect that this study will provide a roadmap for implementation of clinical gene therapy protocols targeting hematopoietic cells for Farber disease and other LSDs.