After two decades of clinical trials, gene therapy demonstrated effectiveness in the treatment of a series of diseases. Currently, several gene therapy products are approved and used in the clinic. Lentiviral vectors (LVs) are one of the most used transfer vehicles to deliver genetic material and the vector of choice to modify hematopoietic cells to correct primary immunodeficiencies, hemoglobinopathies, and leukodystrophies. LVs are also widely used to modify T cells to treat cancers in immunotherapies (e.g., chimeric antigen receptors T cell therapies, CAR‐T). In genome editing, LVs are used to deliver sequence‐specific designer nucleases and DNA templates. The approval LV gene therapy products (e.g., Kymriah, for B‐cell Acute lymphoblastic leukemia treatment; LentiGlobin, for β‐thalassemia treatment) reinforced the need to improve their bioprocess manufacturing. The production has been mostly dependent on transient transfection. Production from stable cell lines facilitate GMP compliant processes, providing an easier scale‐up, reproducibility and cost‐effectiveness. The establishment of stable LV producer cell lines presents, however, several difficulties, with the cytotoxicity of some of the vector proteins being a major challenge. Genome editing technologies pose additional challenges to LV producer cells. Herein the major bottlenecks, recent achievements, and perspectives in the development of LV stable cell lines are revised.

Adeno-associated viral (AAV) vectors represent one of the leading platforms for gene delivery. Nevertheless, their small packaging capacity restricts their use for diseases requiring large-gene delivery. To overcome this, dual-AAV vector systems that rely on protein trans-splicing were developed, with the split-intein Npu DnaE among the most-used. However, the reconstitution efficiency of Npu DnaE is still insufficient, requiring higher vector doses. In this work, two split-inteins, Cfa and Gp41-1, with reportedly superior trans-splicing were evaluated in comparison with Npu DnaE by transient transfections and dual-AAV in vitro co-transductions. Both Cfa and Gp41-1 split-inteins enabled reconstitution rates that were over two-fold higher than Npu DnaE and 100% of protein reconstitution. The impact of different vector preparation qualities in split-intein performances was also evaluated in co-transduction assays. Higher-quality preparations increased split-inteins’ performances by three-fold when compared to low-quality preparations (60–75% vs. 20–30% full particles, respectively). Low-quality vector preparations were observed to limit split-gene reconstitutions by inhibiting co-transduction. We show that combining superior split-inteins with higher-quality vector preparations allowed vector doses to be decreased while maintaining high trans-splicing rates. These results show the potential of more-efficient protein-trans-splicing strategies in dual-AAV vector co-transduction, allowing the extension of its use to the delivery of larger therapeutic genes.

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 gag-pol and rev cassettes, expressed under the control of constitutive promoters, showed robust expression generating titers of 10⁹ physical particles (P.P.s)/mL. However, Pol 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 trans-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.