Generation of High-Titer Self-Inactivated γ-Retroviral Vector Producer Cells

Abstract

The γ-retroviral vector is a gene delivery vehicle that is commonly used in gene therapy. Despite its efficacy, its strong enhancers contributed to malignant transformations in some hematopoietic stem cell (HSC) gene therapy trials. A safer version without viral enhancers (SIN) is available, but its production is cumbersome, as high titers can only be obtained in transient transfection. Our aim was to develop a system that could easily generate high-titer SIN vectors from stable producer cells. The use of the cytomegalovirus enhancer-promoter sequence to generate the full-length genomic RNA combined to sequences that decrease transcriptional readthrough (WPRE and strong polyadenylation sequences) led to 6 × 10 6 infectious units (IU)/mL of a SIN GFP vector in transient transfection. The incorporation of a blasticidin selection cassette to the retroviral plasmid allowed the generation of stable clones in the 293Vec packaging cells that release 2 × 10 7 IU/mL and 1.4 × 10 7 IU/mL of a SIN GFP and a SIN PIGA vector, respectively. A titer of 1.8 × 10 6 IU/mL was obtained with a SIN vector containing the long 8.9-kb COL7A1 cDNA. Thus, an efficient process was established for the generation of stable 293Vec-derived retrovirus producer cells that release high-titer SIN vectors.

Non-invasive Reporter Gene Imaging of Cell Therapies, including T Cells and Stem Cells

“…differentiated from stem cells). In contrast, γ-retroviruses efficiently transduce only actively dividing cells, and have been commercially approved for use in gene therapy applications for ex vivo modification of T-cells and haematopoietic stem cells [34]. In some cases,.…”

Tunable metal–organic frameworks assist in catalyzing DNAzymes with amplification platforms for biomedical applications

“…221,222 However, due to the uncertainty of in vivo editing, many random integration or off-target events cannot be avoided, and some viral vectors will also cause lifethreatening immune responses. [223][224][225] Therefore, the development and optimization of novel gene delivery vector systems so that therapeutic genes can be more safely and efficiently delivered to targeted cells or tissues will greatly promote the process of gene therapy in clinical applications. Whether it is in vivo or in vitro gene therapy, the selection of ideal vectors is the key to the success of gene therapy.…”

HEK293 Cell Line as a Platform to Produce Recombinant Proteins and Viral Vectors

“…Stable, suspension-adapted producer cell lines developed from packaging cell lines could produce yRVs in a serum-free medium have been developed to improve process scalability ( Ghani et al, 2007 ). Subsequently, yRVs producer cell lines for the safer, self-inactivating (SIN) yRVs have also been developed ( Loew et al, 2010 ; Ghani et al, 2019 ). SIN vectors were first developed by the modification of the U3 sequences of the 3 viral LTR, resulting in the loss of viral enhancer and promoter sequences upon integration in the transduced cell ( Yu et al, 1986 ) This reduces the risk of RCV formation as the viral promoter and enhancers are absent, thus preventing vector mobilisation.…”