Transgenic HIV-1-derived lentiviral particles are at the forefront of current gene therapy and tissue engineering initiatives, which will require optimal protocols for large-scale production of clinical-grade therapeutic lentiviruses. Production of latest-generation self-inactivating lentiviral particles requires cotransfection of mammalian production cell lines with two helper plasmids along with the lentivector, whose transgene-encoding expression cassette is the only genetic information stably transduced into target chromosomes. Capitalizing on a recently designed lentiviral expression vector family, we conducted rigorous analysis of production-relevant parameters including transfection, cell density, media composition, temperature, relative (helper) vector concentrations and genetic configuration. Comparative analysis of lentiviral particle performance (VP) was based on the viral titer (reflecting the number of transduction-competent lentiviral particles) relative to the number of lentiviral particles produced (correlating with p24 production levels) (VP=titer/viral particle number). Optimal lentiviral production parameters, resulting in up to 132-fold greater VP compared to standard protocols, required (i) CaPO4-based transfection (ii) of helper plasmids and lentivector at a fixed concentration ratio (helper plasmid I:helper plasmid II:lentivector=1:1:2) (iii) into 1x10(5) human embryonic kidney cells/cm2 (HEK293-T) (iv) cultivated at 37 degrees C (v) in Advanced D-MEM medium supplemented with (vi) 2% fetal calf serum, (vii) and a culture additive containing 0.01 mM cholesterol, 0.01 mM egg's lecithin and 1x chemically defined lipid concentrate. (viii) Furthermore, constitutive transgene expression units placed in a forward polyadenylation site (pA)-free orientation relative to the lentivector backbone resulted in optimal transgene transduction/expression. Our studies suggest that detailed knowledge of lentivector design and the production of lentiviral particles will advance large-scale manufacturing of clinically relevant lentiviruses for future gene therapy applications.