The impact of lentiviral vector genome size and producer cell genomic to gag-pol mRNA ratios on packaging efficiency and titre

Sweeney, Nathan P.; Vink, Conrad A.

doi:10.1016/j.omtm.2021.04.007

Cited by 48 publications

(31 citation statements)

References 36 publications

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“…S alone was most effective in generating a mixed population of iOPCs (see below). In agreement with previous studies [ 36 , 37 ], we observed a higher level of expression of smaller-sized constructs. However, an intermediate selection step performed in our protocol normalized these differences and allowed us to compare TF combinations irrespective of construct size.…”

Section: Discussionsupporting

confidence: 93%

Expression of Lineage Transcription Factors Identifies Differences in Transition States of Induced Human Oligodendrocyte Differentiation

Raabe

Stephan

Waldeck

et al. 2022

Cells

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Oligodendrocytes (OLs) are critical for myelination and are implicated in several brain disorders. Directed differentiation of human-induced OLs (iOLs) from pluripotent stem cells can be achieved by forced expression of different combinations of the transcription factors SOX10 (S), OLIG2 (O), and NKX6.2 (N). Here, we applied quantitative image analysis and single-cell transcriptomics to compare different transcription factor (TF) combinations for their efficacy towards robust OL lineage conversion. Compared with S alone, the combination of SON increases the number of iOLs and generates iOLs with a more complex morphology and higher expression levels of myelin-marker genes. RNA velocity analysis of individual cells reveals that S generates a population of oligodendrocyte-precursor cells (OPCs) that appear to be more immature than those generated by SON and to display distinct molecular properties. Our work highlights that TFs for generating iOPCs or iOLs should be chosen depending on the intended application or research question, and that SON might be beneficial to study more mature iOLs while S might be better suited to investigate iOPC biology.

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Section: Discussionsupporting

confidence: 93%

Expression of Lineage Transcription Factors Identifies Differences in Transition States of Induced Human Oligodendrocyte Differentiation

Raabe

Stephan

Waldeck

et al. 2022

Cells

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“…A size reduction of 1 kb improved the transduction e ciency by more than 5-fold, which is consistent with previous work [21][22][23][24]. The two smallest CRISPR-Cas systems CjCas9 and RfxCas13d did yield the highest transduction titer, and the largest SpCas9 and AsCas12a systems exhibit the lowest titer.…”

Section: Production Is Not Affected By Increasing Size Of the Transge...supporting

confidence: 89%

“…Whereas the LV transduction e ciency on human and mouse stem cells dropped dramatically for viral RNAs that approached 6 kb, a modest reduction of their size of 0.6 kb rescued the transduction e ciency by more than threefold. Consistently, Sweeney et al showed that the LV titer drops with increasing genome size [24]. In general, genome sizes close to or exceeding that of the natural HIV template (9.8 kb) result in decreased packaging capacity and consequently a low transduction e ciency.…”

Section: Introductionmentioning

confidence: 82%

“…This value could be a rough estimate of the upper size of RNA transcripts that can be packaged, albeit rather ine ciently, in a LV particle. It is indeed likely that a reduced packaging e ciency is witnessed for RNA genome sizes that exceed the RNA genome size of the HIV pathogen (9.8 kb) [24].…”

Section: Production Is Not Affected By Increasing Size Of the Transge...mentioning

confidence: 99%

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Impact of the size of CRISPR-Cas gene cassettes on the packaging efficiency and transduction titer of lentiviral vectors

Fan

Berkhout

Herrera-Carrillo

2022

Preprint

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BackgroundCRISPR-Cas technology has revolutionized the use of genome-editing in studies on diverse biological topics. Lentiviral vectors (LVs) have been widely used for the generation of stably transduced cells that durably express the Cas protein and guide RNA (gRNA), the key components of the CRISPR-Cas editing system. This delivery system allows sustained gene expression in dividing and non-dividing cells in vitro and in vivo and LVs can be pseudo-typed with a variety of envelope proteins to establish a broad tropism for a range of target cells. A major challenge on the use of LVs in clinical settings is the requirement to obtain vector stocks of a sufficiently high titer. The endonuclease encoded by Streptococcus pyogenes Cas9 (SpCas9) is the most widely used CRISPR endonuclease for genome engineering, but it has an important limitation because of its relatively large size (∼4.1 kb coding sequence) that may hamper the delivery by means of viral vector systems. Smaller Cas endonucleases have been developed more recently, but these systems still need to be validated in specific experimental settings.ResultsThis work aimed to assess the impact of the CRISPR-Cas transgene size on the production of LVs and their ability to transduce cells. We observed that the LV transduction efficiency measured on the SupT1 T cell line dropped dramatically for RNA transcripts approaching 8 kb, e.g. a further increase of 1 kb reduced the transduction efficiency by more than 5-fold. As the number of produced virus particles was not affected, this means that the use of larger transgenes may cause the production of more empty virion particles. ConclusionsThis study confirmed that the transfer efficiency decreased for CRISPR-Cas genes above a certain size. One of the strategies to avoid the restricted RNA packaging can be the use of smaller CRISPR-Cas systems or the inclusion of truncated versions of the regulatory sequences like the promoters that are needed for efficient expression of the transgenes. This work has implications for the design of effective CRISPR-Cas editing therapies that use viral vectors, LVs in particular.

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“…Large transgenes exceeding 10 kb have been reported [60,61]. However, the accommodation of large transgenes may come at the cost of lower functional viral titers, as previous studies showed a semi-logarithmic reduction of 3 to 4 folds in functional LV titer per kb increase in genome size [60,62]. Aside from these major advantages, LVs are negligibly inflammatory and induce only minute levels of phenotypic and functional DC maturation in vitro and in vivo [63] and our unpublished data.…”

Section: Advantages Of Lvs As Vaccine Vectorsmentioning

confidence: 61%

Use of lentiviral vectors in vaccination

Charneau

Majlessi

2021

Expert Review of Vaccines

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Introduction: Lentiviral vectors have emerged as powerful vectors for vaccination, due to their high efficiency to transduce dendritic cells and to induce long-lasting humoral immunity, CD8 + T cells, and effective protection in numerous preclinical animal models of infection and oncology.Areas covered: Here, we reviewed the literature, highlighting the relevance of lentiviral vectors in vaccinology. We recapitulated both their virological and immunological aspects of lentiviral vectors. We compared lentiviral vectors to the gold standard viral vaccine vectors, i.e., adenoviral vectors, and updated the latest results in lentiviral vector-based vaccination in preclinical models.Expert opinion: Lentiviral vectors are non-replicative, negligibly inflammatory, and not targets of preexisting immunity in human populations. These are major characteristics to consider in vaccine development. The potential of lentiviral vectors to transduce non-dividing cells, including dendritic cells, is determinant in their strong immunogenicity. Notably, lentiviral vectors can be engineered to target antigen expression to specific host cells. The very weak inflammatory properties of these vectors allow their use in mucosal vaccination, with particular interest in infectious diseases that affect the lungs or brain, including COVID-19. Recent results in various preclinical models have reinforced the interest of these vectors in prophylaxis against infectious diseases and in onco-immunotherapy.

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The impact of lentiviral vector genome size and producer cell genomic to gag-pol mRNA ratios on packaging efficiency and titre

Cited by 48 publications

References 36 publications

Expression of Lineage Transcription Factors Identifies Differences in Transition States of Induced Human Oligodendrocyte Differentiation

Expression of Lineage Transcription Factors Identifies Differences in Transition States of Induced Human Oligodendrocyte Differentiation

Impact of the size of CRISPR-Cas gene cassettes on the packaging efficiency and transduction titer of lentiviral vectors

Use of lentiviral vectors in vaccination

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