US 11,993,781 B2
Stable pseudotyped lentiviral particles and uses thereof
Anne Galy, Fontainebleau (FR)
Assigned to Institut National de la Santé et de la Recherche Médicale, Paris (FR); and Université d'Évry Val d'Éssonne
Appl. No. 16/095,098
Filed by GENETHON, Evry (FR); INSERM (INSTITUT NATIONAL DE LA SANTÉ ET DE LA RECHERCHE MÉDICALE), Paris (FR); and UNIVERSITE D'EVRY VAL D'ESSONNE, Evry (FR)
PCT Filed Apr. 21, 2017, PCT No. PCT/EP2017/059465
§ 371(c)(1), (2) Date Oct. 19, 2018,
PCT Pub. No. WO2017/182607, PCT Pub. Date Oct. 26, 2017.
Claims priority of application No. 16305466 (EP), filed on Apr. 21, 2016.
Prior Publication US 2019/0300902 A1, Oct. 3, 2019
Int. Cl. C12N 15/63 (2006.01); A61K 35/17 (2015.01); A61K 35/76 (2015.01); C07H 21/04 (2006.01); C07K 14/005 (2006.01); C12N 5/0781 (2010.01); C12N 15/85 (2006.01); C12N 15/86 (2006.01); A61K 48/00 (2006.01)
CPC C12N 15/86 (2013.01) [A61K 35/17 (2013.01); A61K 35/76 (2013.01); C07K 14/005 (2013.01); C12N 5/0635 (2013.01); A61K 48/00 (2013.01); C12N 2740/10022 (2013.01); C12N 2740/16022 (2013.01); C12N 2740/16043 (2013.01); C12N 2740/16052 (2013.01)] 5 Claims
 
1. A method for obtaining stable lentiviral particles pseudotyped with an endogenous retroviral syncytin (ERV syncytin) and packaging a heterologous gene of interest, which have a high physical and/or infectious titer(s), comprising the following steps:
a) transfecting four plasmids in appropriate cell lines, wherein said four plasmids comprise a first plasmid comprising the heterologous gene of interest, a second plasmid comprising the lentiviral rev gene, a third plasmid comprising the gag and pol genes, and a fourth plasmid comprising a nucleotide sequence coding for an envelope glycoprotein, wherein the envelope glycoprotein consists of an ERV syncytin selected from the group consisting of HERV-W, HERV-FRD, murine syncytin-A and murine syncytin-B;
b) incubating the transfected cells obtained in a), so that they produce the stable lentiviral particles pseudotyped with an ERV syncytin, and packaging the heterologous gene of interest;
c) harvesting the stable lentiviral particles obtained in b) 24 hours after the transfection, before fusion of the transfected cells, and
d) concentrating the stable lentiviral particles harvested in c) to obtain a preparation of concentrated stable lentiviral particles having a high infectious titer higher than 1×106 TU/mL or a high physical titer higher than 1.5×10 5 ng p24/mL after freezing and thawing of the preparation.