Deciphering key parameters enhancing lentiviral vector producer cells yields: Vector components copy number and expression

The use of lentiviral vectors (LVs) in gene therapy is expanding, demanding high-quality viral preparations. Producer cell lines for LV production offer robust manufacturing platforms. However, their development is still progressing and more knowledge on the impact of vector components expression levels on vector yields and quality is essential. This work studies the impact of vector cassette expression and stability on vector titer and quality, identifying key parameters in cell line development. Ten heterogeneous LV stable producer clones established through random cassette integration were characterized. The <i>gag-pol</i> and <i>rev</i> cassettes, expressed under the control of constitutive promoters, showed robust expression generating titers of 10⁹ physical particles (P.P.s)/mL. However, <i>Pol</i> and reverse transcriptase expressions were shown to be better indicators of potential functional titers. Envelope and transfer vector expression levels were key to attaining high functional particles yields. The stability analysis of two top clones and their <i>trans</i>-complementation with each genetic cassette further supported this conclusion. The producer LV clones expressed constitutively the 4070A envelope, but the overexpression of the VSV-G envelope increased 30-fold the titer supporting the envelope as key determinant in LV quality. This work further elucidates bottlenecks in LV producer cell line development providing insights for their optimization.