Method for determining the loading state of an aav particle by nuclear magnetic resonance relaxometry

Abstract

The current invention is based, at least in part, on the finding that the transverse nuclear magnetic spin relaxation time T2 and the transverse nuclear magnetic spin relaxation rate R2, respectively, of protons of water molecules in an aqueous solution comprising viral particles depends on the loading status (full vs. empty) of the viral particle. Thus, one aspect of the current invention is a method for determining the ratio of loaded viral particles to empty viral particles in a sample, comprising the steps of determining a nuclear magnetic resonance (NMR) parameter related to the protons of the water molecules present in an aqueous solution comprising a mixture of loaded and empty viral particles by applying an NMR measurement to the solution, and determining the ratio of loaded viral particles to empty viral particles with the NMR parameter determined in the previous step based on a calibration function.

1 . A method for determining a ratio of DNA-containing AAV particles to empty AAV particles in a sample, comprising the following steps:
 determining a nuclear magnetic resonance (NMR) parameter of the protons of the water molecules in the sample, wherein the sample is an aqueous solution comprising a mixture of DNA-containing AAV particles and empty AAV particles, by applying an NMR measurement to the solution, and   determining the ratio of DNA-containing AAV particles to empty AAV particles with the determined NMR parameter using a calibration function.   
     
     
         2 . The method according to  claim 1 , wherein the NMR parameter is a transverse nuclear magnetic spin relaxation time T 2  or a transverse nuclear magnetic spin relaxation rate R 2 , of the protons of the water molecules in the aqueous solution. 
     
     
         3 . The method according to  claim 2 , wherein a magnetic field strength during the determination of the NMR parameter is about 0.47 T. 
     
     
         4 . The method according to  claim 3 , wherein a resonance frequency during the determination of the NMR parameter is about 20 MHz. 
     
     
         5 . The method according to  claim 4 , wherein the AAV particle is of an AAV2 serotype, or an AAV6 serotype, or an AAV8 serotype. 
     
     
         6 . The method according to  claim 5 , wherein the calibration function is a second order polynomial function. 
     
     
         7 . The method according to  claim 6 , wherein the calibration function is obtained by determining the same NMR parameter for at least three calibration samples, the first calibration sample comprising DNA-containing AAV particles and empty AAV particles at a ratio in the range of 0:100 to 30:70, the second calibration sample comprising DNA-containing AAV particles and empty AAV particles at a ratio in the range of 40:60 to 60:40, and the third calibration sample comprising DNA-containing AAV particles and empty AAV particles at a ratio in the range of 70:30 to 100:00. 
     
     
         8 . The method according to  claim 7 , wherein the total concentration of AAV particles is at least 1*10 12  vp/mL. 
     
     
         9 . The method according to  claim 8 , wherein the method is an in vitro method. 
     
     
         10 . Use of a transverse nuclear magnetic spin relaxation time T 2  for a determination of a loading status of AAV particles. 
     
     
         11 . The use according to  claim 10 , wherein the loading status is a ratio of DNA-containing AAV particles to empty AAV particles. 
     
     
         12 . The use according to  claim 10 , wherein the loading status is a fraction of DNA-containing AAV particles. 
     
     
         13 . The use according to  claim 10 , wherein the loading status is a concentration of DNA-containing AAV particles. 
     
     
         14 . The use according to  claim 10 , wherein the loading status is determined in a sample. 
     
     
         15 . The use according to  claim 10 , wherein the determination is in an aqueous solution and the transverse nuclear magnetic spin relaxation time T 2  is the transverse nuclear magnetic spin relaxation time T 2  of the protons of the water molecules in the aqueous solution.