Optimized production and concentration of lentiviral vectors containing large inserts

Abstract

Generation of high titer lentiviral stocks and efficient virus concentration are central to maximize the utility of lentiviral technology. Here we evaluate published protocols for lentivirus production on a range of transfer vectors differing in size (7.5-13.2 kb). We present a modified virus production protocol robustly yielding useful titers (up to 10 7 /ml) for a range of different transfer vectors containing packaging inserts up to 7.5 kb. Moreover, we find that virus recovery after concentration by ultracentrifugation depends on the size of the packaged inserts, heavily decreasing for large packaged inserts. We describe a fast (4 h) centrifugation protocol at reduced speed allowing high virus recovery even for large and fragile lentivirus vectors. The protocols outlined in the current report should be useful for many labs interested in producing and concentrating high titer lentiviral stocks.

Gene Therapy Strategies Incorporating Large Transgenes

“…Although still constrained by encapsidation limitations, lentiviruses can be used to shuttle a number of transgenes that are restricted within the adeno-associated viral vector. However, as the insert size increases the viral particles produced (as measured by viral titer) can decrease (Yacoub et al, 2007). This phenomenon has been observed by several laboratories that use the 4.5 kb B domain deleted FVIII transgene.…”

“…Further cleavage occurs extracellularly in which thrombin cleavage releases the remainder of the B domain resulting in FVIII activation (Lenting et al, 1998). Large transgenes, such as the FVIII transgene, complicate gene therapy applications using viral vectors by (1) limiting the types of vectors available due to encapsidation limitations (see Table I) and (2) reducing the titer of viral vector that can be produced (Kumar et al, 2001;Radcliffe et al, 2008;Yacoub et al, 2007). The different human cDNA transgene lengths chosen for the clinical trials are a reflection of these limitations.…”

“…Although retroviruses have the capability of encapsulating 7kb of exogenous DNA, incorporating a 7kb transgene into a retrovirus results in diminished viral titer making it difficult to achieve a high enough number of viral particles to be effective. Reducing the size of the insert, however, can increase viral titer (Yacoub et al, 2007). For this reason, many groups are using viral vectors and have reduced the size of the cDNA to approximately 4.5 kb.…”

CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes

“…Lentiviruses are able to infect non-dividing cells and have been used in vivo to efficiently transduce a variety of specific target organs (Cockrell and Kafri, 2007). Furthermore, the packaging limit of lentivirus is ~8.5 kb (although inserts larger than ~3 kb are packaged less efficiently), sufficient to package most Cas9 genes, guide RNA expression constructs, and required promoter and regulatory sequences (Kumar et al, 2001;Yacoub et al, 2007). Lentiviruses have been successfully used to deliver Cas9 and sgRNA genes into mice to characterize the contributions of a panel of tumor suppressor genes to the progression of lung cancer (Sanchez-Rivera et al, 2014).…”

Programming Human Pluripotent Stem Cells into White and Brown Adipocytes

“…The vectors-either lenti-rtTA plasmid [50][51][52][53] or lenti-PPARG2 plasmid-together with the two packaging plasmids-pMDL, pREV-and the plasmid coding for VSV-G envelope were transfected into HEK293T cells using calcium chlorate as previously described 54 Cell media/viral supernatant was collected 48 and 72 h after transfection, passed through a 0.45 μm filter and used directly for the transduction of cells. We determined the RNA copy numbers per millilitre of viral supernatant using the lenti-X RT-qPCR titration kit.…”