LYOPHILIZED PLASMID/DNA TRANSFECTION REAGENT CARRIER COMPLEX

• US 20110008894A1
• Filed: 07/06/2010
• Published: 01/13/2011
• Est. Priority Date: 07/07/2009
• Status: Abandoned Application

• Current Assignee: CAYLA
• Original Assignee: CAYLA
• Inventors: Vernejoul, Fabienne, Reynes, Jean-Paul, Drocourt, Daniel, Tiraby, Gerard, Perouzel, Eric, RAVET, Emmanuel
• Application Number:
  US12/830,614
• Publication Number:
  US 20110008894A1
• Time in Patent Office: Days
• US Class Current: 435/455
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle
• CPC Class Codes: C12N 2740/15043 viral genome or elements thereof as genetic vector

Abstract

Disclosed is a novel formulation for the production of a lyophilized plasmid/DNA transfection reagent complex capable of serving as a carrier for additional free plasmids. Upon rehydration, this plasmid/DNA transfection reagent carrier can be used to introduce simultaneously the complexed plasmid and the additional free plasmids into animal cells. This novel formulation can be useful for viral particle production, gene transfer experiments like gene silencing experiments, reporter gene, or integration/selection experiments.

Claims

1. A method for transfecting an animal cell, comprising the steps of:

a) rehydrating a lyophilized composition comprising a lyoprotectant and one or more plasmids complexed with a DNA transfection reagent;

b) adding to the rehydrated composition obtained in step a) one or more free plasmids; and

c) transfecting an animal cell with the mixture obtained in step b).

2. The method of claim 1, wherein the transfection reagent comprises a cationic polymer or a cationic lipid with or without a neutral helper lipid.

3. The method of claim 2, wherein the transfection reagent comprises a cationic lipid with a neutral helper lipid.

4. The method of claim 2, wherein the transfection reagent comprises a cationic polymer.

5. The method of claim 1, wherein the lyoprotectant is a carbohydrate.

6. The method of claim 1, wherein the lyophilized composition further comprises a salt.

7. The method of claim 1, wherein said lyophilized composition comprises two or more plasmids complexed with a DNA transfection reagent:

a packaging plasmid coding for a pseudotyping gene, and

one or more plasmids coding for one or more lentiviral accessory genes selected from the group consisting of gag, tat, pol and rev.

8. The method of claim 1, wherein lyophilized composition comprises three plasmids complexed with a DNA transfection reagent:

a plasmid coding for one or more lentiviral accessory genes selected from the group consisting of gag, tat and pol;

a plasmid coding for rev, and

a packaging plasmid coding for a pseudotyping gene.

9. The method of claim 1, wherein the one or more additional free plasmids added in step b) encode a protein or a RNA of interest.

10. A kit comprising:

a) a lyophilized composition comprising a lyoprotectant and one or more plasmids complexed with a DNA transfection reagent, and

b) an expression vector comprising a cloning site which enables the introduction of a nucleotide of interest.