RETROVIRAL PARTICLE COMPRISING AT LEAST TWO ENCAPSIDATED NONVIRAL RNAS
The present invention relates to a retro viral system for the transfer of non-viral RNA into target cells and more particularly a retroviral particle capable of delivering multiple RNAs. More particularly, it relates to retroviral particles comprising a protein derived from the Gag polyprotein, an envelope protein, optionally an integrase and at least two encapsidated non-viral RNAs, the encapsidated non-viral RNAs each comprising an RNA sequence of interest linked to an encapsidation sequence, each encapsidation sequence being recognised by a binding domain introduced into the protein derived from the Gag polyprotein and/or into the integrase.
1 . A method of manufacturing a retroviral particle, comprising a step of co-transfecting producer cells with:
(i) an expression plasmid comprising DNA coding for at least two different, non-viral RNA sequences, each RNA sequence comprising a sequence of interest linked to an encapsidation sequence, wherein the encapsidation sequence is either upstream or downstream of each of the sequence of interest,
(ii) an encapsidation plasmid coding for a protein from a Gag polyprotein and optionally an integrase, said protein and/or integrase being chimeric as it comprises an heterologous binding domain enabling each encapsidation sequence to be recognized, and
(iii) an envelope plasmid coding for an envelope protein,
culturing the transfected cells to produce a supernatant comprising the particle and harvesting the supernatant thus manufacturing the particle.
2 . A method of manufacturing a retroviral particle, comprising a step of co-transfecting producer cells with:
(i) an expression plasmid comprising DNA coding for at least two different, non-viral RNA sequences, each RNA sequence comprising a sequence of interest linked to an encapsidation sequence, wherein the encapsidation sequence is either upstream or downstream of each of the sequence of interest, or alternatively a first and a second expression plasmid each comprising DNA coding for one of at least two different, non-viral RNA sequences, each RNA sequence comprising a sequence of interest linked to an encapsidation sequence, wherein the encapsidation sequence is either upstream or downstream of each of the sequence of interest,
(ii) an encapsidation plasmid coding for a nucleocapsid protein, which is chimeric, as it comprises an heterologous binding domain making it possible to recognize each encapsidation sequence, and
(iii) an envelope plasmid coding for an envelope protein,
culturing the transfected cells to produce a supernatant comprising the particle and harvesting the supernatant thus manufacturing the particle.
3 . The method of manufacturing the particle according to claim 2 ,
wherein the sequences of interest are different and the encapsidation sequences are identical.
4 . The method of manufacturing the particle according to claim 1 , further comprising a step of clarifying the supernatant from the cell culture of the transfected cells.
5 . The method of manufacturing the particle according to claim 4 , further comprising a step of concentrating and/or purifying the supernatant from the cell culture of the transfected cells.
6 . In a method for transducing cells with a retroviral particle, comprising transducing cells in vitro or ex vivo with a retroviral particle, the improvement wherein the retroviral particle is the retroviral particle of claim 1 .