Kinetics of Expression

Lentiviral vectors: Kinetics of Expression

Multiple mutants are available as options, issued from GEG Tech R&D team innovations. The modification of some features of lentiviral vectors enlarges their scope of applications.

Integration-Deficient Lentiviral Vectors (mutations D64V, N and LQ) lead to the generation of episomal vector genomes. They can be used for transient expression in dividing cells or long term expression in non-dividing cells without the limits inherent to the integration of the vector.

Reverse Transcription-Deficient Lentiviral Vectors result from the mutation of the reverse-transcriptase (mutations D110E, E478Q and D110E/E478Q), which leads to the production of a vector whose genome cannot be reverse-transcribed. Consequently, there is no DNA synthesis and the RNA vector genome is taken in charge by the transduced cell machinery like a mRNA. Those vectors allow a fully transient expression regardless of the type of cells (dividing or not).

When applicable, reducing the transduction volume allows increased transduction efficiency without increasing the vector dose and possibly related toxicity.

Kinetics of Expression -

This graph schematizes the kinetics of expression observed for each kind of lentiviral vector:

  • Integrative LV expression is high and stable over months

  • IDLV expression in non-dividing cells is also stable for months, yet a bit lower

  • IDLV expression in dividing cells is short-term and depends on the dividing rate of the cells

  • RTDLV expression is transient for all kinds of cells, and lower

Integrating Lentiviral Vectors

They are classic lentiviral vectors, derived from HIV-1 and deficient for replication. They are produced with an integration proficient integrase, allowing their DNA genome to be stably integrated into the host cell chromatin. They allow robust and long term expression in dividing and non-dividing cells. Their tropism is defined by the envelop glycoprotein they have been produced with.

Integration of the transgene with a suitable promoter allows robust and long term expression, even after cell divisions. Integration is expected to be favored in transcriptionally active regions of the chromatin and may induce genotoxicity such as deregulation of the transcriptome of transduced cells, potentially inducing immortalization of cells. GEG Tech offers Lenti-ONE™ with a mutant integrase expected to display an integration profile closer to a random distribution and reduced genotoxicity. Lenti-ONE™ with IN(R166A) or an IN(Q168A) integrase mutant are hampered for interaction with LEDGF, a cellular factor, and yet retain integration capability.

Integrating Lentiviral Vectors

IDLV are derived from HIV-1 and are deficient for integration. Hence, the DNA genomes of these lentiviral vectors are non-integrative and remain in the transduced cell nucleus as DNA episomes, which are diluted over the cell divisions. In contrast in non-dividing cells, episomals forms are degraded very slowly and allow long term expression of the transgene, observed up to several weeks in vitro and several months in vivo. Such vectors are of particular interest when willing to have low integration frequency per cell, for instance when developing cell lines or for transgenesis purposes.

The modifications of integrase consist of two classes of integrase mutants: catalytic domain mutant D64V and C-terminal basic domain mutants N or LQ. They are used for transient expression in dividing cells and long term expression in non-dividing cells with reduced genotoxicity as compared to integrative vectors.

Three mutations are available. Residual integration can be observed and is inherent to the presence of a DNA molecule, which is able to recombine with host cell chromatin. The background integration level ranges between 0.5 and 1% and depends on the integrase mutation. The integrase mutation also seems to influence the transgene expression level:

  • the D64V integrase shows the highest level of expression and the highest integration background

  • The N integrase shows the lowest level of expression and the lowest integration background

  • The LQ integrase shows intermediate level of expression and integration background

Consequently, the choice of the integrase depends on your experimental context.

Reverse Transcription-Deficient Lentiviral Vectors

They also are derived from HIV-1, but are deficient for reverse transcription. The RNA vector genome is thus taken in charge by the host cell machinery as an mRNA and directly translated into a protein. Consequently, there is no DNA form of the genome involved. They are used to mediate fully transient expression in dividing and non-dividing cells. The reverse transcription blockade results from one or two mutations of the reverse transcriptase which is necessary to translate the RNA genomes into DNA. The mutations of the reverse transcriptase that we use completely block the reverse transcription step, consequently, no DNA and residual integration is observed (residual integration is close to 0.1%).

GEG Tech provides three different reverse transcriptase mutants: D110E, R178Q and the double mutant D110E/R178Q. Through our experimental results, we didn’t point out a significant difference of efficiency or background of reverse transcriptase activity between these three vectors.

The level of transgene expression is also reduced compared to other LV. Consequently, RTDLV are particularly suited for transient expression and when the level of expression required is low:

  • Expression of genome editing tools

  • Expression of differentiation factors
Kinetics of Expression -

WT: Reverse Transcription and integration steps are completed leading to integration into host cell chromatin of a double stranded DNA lentiviral genome.

IDLV: The integration step is blocked so that the double stranded DNA lentiviral genome resulting from Reverse Transcription remains episomal.

RTDLV: Reverse Transcription is blocked so that the single stranded RNA lentiviral genome delivered by the particle is taken over by cell machinery as a mRNA and is directly translated in protein.