Lentiviral vector packaging and producer cell lines yield titers equivalent to the industry-standard four-plasmid process.

Lentiviral vector (LVV)-mediated cell and gene therapies have the potential to cure diseases that currently require lifelong intervention. However, the requirement for plasmid transfection hinders large-scale LVV manufacture. Moreover, large-scale plasmid production, testing, and transfection contribute to operational risk and the high cost associated with this therapeutic modality.

Improved GaLV-TR Glycoproteins to Pseudotype Lentiviral Vectors: Impact of Viral Protease Activity in the Production of LV Pseudotypes.

Lentiviral vectors (LVs) are excellent tools for gene transfer into mammalian cells. It is noteworthy that the first gene therapy treatment using LVs was approved for commercialization in 2017. The G glycoprotein from rhabdovirus vesicular stomatitis virus (VSV-G) is the glycoprotein most used to pseudotype LVs, due to its high efficiency in transducing several cell types and its resistance to viral vector purification and storage conditions.

Pseudotyping retrovirus like particles vaccine candidates with Hepatitis C virus envelope protein E2 requires the cellular expression of CD81.

Hepatitis C virus (HCV) infects 3% of world population being responsible for nearly half a million deaths annually urging the need for a prophylactic vaccine. Retrovirus like particles are commonly used scaffolds for antigens presentation being the core of diverse vaccine candidates. The immunogenicity of host proteins naturally incorporated in retrovirus was hypothesized to impact the performance of retrovirus based vaccines.

Flexible pseudotyping of retrovirus using recombinase-mediated cassette exchange.

Objective: Develop an engineered cell line containing two flexible gene expression systems enabling the continuous production of tailor-made recombinant gammaretrovirus with predictable productivities through targeted integration.

Results: Dual-FLEX cells (dFLEX) contain two independent recombinase-mediated cassette exchange (RMCE) systems which confer flexibility to the expression of different transgene and envelope combinations. The flexible envelope expression in dFLEX cells was validated by pseudotyping retrovirus particles with three different viral envelope proteins-GaLV, 4070A and VSV-G.

Disclosing the Parameters Leading to High Productivity of Retroviral Producer Cells Lines: Evaluating Random Versus Targeted Integration.

Gammaretrovirus and lentivirus are the preferred viral vectors to genetically modify T and natural killer cells to be used in immune cell therapies. The transduction efficiency of hematopoietic and T cells is more efficient using gibbon ape leukemia virus (GaLV) pseudotyping. In this context gammaretroviral vector producer cells offer competitive higher titers than transient lentiviral vectors productions.

Human carboxylesterase 2: Studies on the role of glycosylation for enzymatic activity.

Human carboxylesterase 2 (hCES2) is a glycoprotein involved in the metabolism of drugs and several environmental xenobiotics, whose crystallization has been proved to be a challenging task. This limitation could partly be due to glycosylation heterogeneity and has delayed the disclosure of the 3D structure of hCES2 which would be of upmost relevance for the development of new substrates and inhibitors. The present work evaluated the involvement of glycans in hCES2 activity and thermo stability in an attempt to find alternative active forms of the enzyme that might be adequate for structure elucidation.

Impact of E1 and Cre on adenovirus vector amplification: developing MDCK CAV-2-E1 and E1-Cre transcomplementing cell lines.

Adenovirus vectors have been extensively studied through the manipulation of viral genome. However, little attention is being paid to their producer cell-lines; cells are selected according to virus yields, neglecting the expression profile of transcomplementing gene products underlying cell performance. This work evaluates the impact of E1 (E1A and E1B) and Cre recombinase levels in the production of E1-deleted and helper-dependent canine adenovirus type 2 (CAV-2) vectors using MDCK cells.