Lenti-ONE™ Epi are derived from HIV-1 and are deficient for replication and 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. Lenti-ONE™ Epi are derived from Lenti-ONE™ by either modification of the integrase or modification of the LTR sequence of the genome. Modification of the LTR sequence reduces integration frequency by approximately 3 folds. 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.
Modification of the integrase we propose consists of two classes of integrase mutants: catalytic domain mutant D64V and C-terminal basic domain mutants N or LQ. These Lenti-ONE™ Epi are used for transient expression in dividing cells and long term expression in non-dividing cells with reduced genotoxicity as compared to LentiVectors.
Lenti-ONE™ Epi tropism is defined by the envelop glycoprotein they have been produced with. GEG Tech offers Lenti-ONE™ Epi pseudotyped with VSV glycoprotein, conferring a broad tropism, and Lenti-ONE™ Epi pseudotyped with Mokola glycoprotein, conferring a glial specific tropism. Depending on your purpose and target cells, Lenti-ONE™ Epi are available at High Titer (107 TU/mL) and High Titer+ (108 TU/mL).
We propose three types of mutated integrases which prevent integration of the LV genome into the host cell chromatin. However 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. To summarize, 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.
Lenti-ONE™ Epi genomes are episomal, i.e. not integrated into the chromatin of transduced cells. Consequently, episomes are diluted out during cell division and the transgene expression is transient. 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.
Lenti-ONE™ Epi are very flexible gene transfer tools and can transduce a large variety of cells. Lenti-ONE™ Epi can induce ubiquitous or specific expression depending on the envelope and the promoter selected for your vector. Indeed the envelope choice will affect the entry mechanism of the vector into the cell and the promoter choice will affect the mechanism of transgene transcription. For example, the VSV envelope allows the entry of the Lenti-ONE™ Epi in almost all mammalian cells and CMV or PGK promoter induces ubiquitous expression of transgenes. Consequently, the combination of these elements produced ubiquitous Lenti-ONE™ Epi. In contrast, a Mokola envelope restricts vector entry into glial cells and the GFAP promoter is specific of this cell type. Consequently, one of these two elements is sufficient to induce glial cells specific expression. If the experimental context allows it, it is also possible to combine a specific envelope with specific promoter to enhance specificity of expression.
Lenti-ONE™ Epi can be used in a large array of experimentations. Here are some examples:
in vitro and in vivo applications:
in vitro or in vivo modeling applications: