Prostate cancer (CaP) is the second leading cause of cancer related deaths in men in the United States. Currently, androgen depletion is an essential strategy for CaP combined with surgery, chemotherapy and radiation. Hormone independent cancer stem cells escaping conventional therapy present a major therapeutic challenge. The available treatment regimens for hormone resistant CaP are only palliative and marginally increase survival. Therefore, novel strategies to eradicate CaP including stem cells are imperative. Oncolytic virus (OV) therapy is a novel approach that overcomes the limitations posed by radiation and chemotherapy. Oncolytic virotherapy of cancer is based on the use of replication competent, tumor selective viruses with limited toxicity. Newcastle Disease Virus (NDV), an avian paramyxovirus, is a safe and promising OV successfully used in many clinical trials. NDV is inherently tumor selective and cytotoxic but replication restricted in normal cells. But, systemically delivered NDV fails to reach solid tumors in therapeutic concentrations and also spreads poorly within the tumors due to barriers including complement, innate immunity and extracellular matrix. Overcoming these hurdles is paramount to realize the exceptional oncolytic efficacy of NDV. Therefore, we engineered the fusion (F) glycoprotein of NDV and generated a recombinant NDV (rNDV) cleavable exclusively by prostate specific antigen (PSA). The rNDV replicated efficiently and specifically only in prostate cancer (CaP) cells but failed to replicate in the absence of PSA. Further, PSA-cleavable rNDV caused specific lysis of androgen independent and dependent/responsive CaP cells with a mean effective concentration (EC50) ranging from 0.01 to 0.1 multiplicity of infection (MOI). PSA retargeted rNDV efficiently lysed three-dimensional prostaspheres, suggesting efficacy in vivo. Also, PSA-cleavable NDV failed to replicate in chicken embryos, indicating absence of pathogenicity to its natural host, chickens. Prostaspheres generated from DU-145 CaP cell line derived xenografts showed self-renewal, proliferative and clonogenic potential in vitro, and exhibited increased tumorigenicity in vivo. Embryonic stem and progenitor cell markers like Nanog, Nestin and CD44 were overexpressed in spheres as compared to the cell line suggesting prostaspheres comprise tumor-initiating cells from CaP. Xenograft and cell line derived prostaspheres were permissive for rNDV replication, when the fusion protein was activated by exogenous PSA. The EC50 against tumor initiating cells was 0.11-0.14 MOI, suggesting an excellent therapeutic margin for in vivo studies. PSA retargeting is likely to enhance the therapeutic index of rNDV owing to tumor restricted replication and enhanced fusogenicity. Our results suggest PSA retargeted rNDV selectively replicates and lyse PSA producing CaP cells including tumor-initiating cells and is a promising candidate for immediate Phase I/II clinical trials. / Ph. D.