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.

Claims

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.sub.2 or a transverse nuclear magnetic spin relaxation rate R.sub.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.sup.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.sub.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.sub.2 is the transverse nuclear magnetic spin relaxation time T.sub.2 of the protons of the water molecules in the aqueous solution.

Description

DESCRIPTION OF THE FIGURES

[0173] FIG. 1 Dependency of T.sub.2 on the DNA-loading of AAV particles of the serotype 2 (AAV2).

[0174] FIG. 2 Dependency of T.sub.2 on the DNA-loading of AAV particles of the serotype 6 (AAV6).

[0175] FIG. 3 Dependency of T.sub.2 on the DNA-loading of AAV particles of the serotype 8 (AAV8)

EXAMPLES

[0176] Materials and Methods

[0177] AAV Particles

[0178] DNA-containing AAV particles as well as empty AAV particles of different serotypes were purchased from Virovek, Hayward, Calif., United States of America, at a concentration of 2*10.sup.13 vg/mL. The concentration of vg/mL (virus genomes per mL) equals the concentration of vp/mL (virus particles per mL) as one genome is packaged in one particle. The DNA of the DNA-containing AAV particles comprised an expression cassette for green fluorescent protein with a CMV promoter (CMV-GFP):

[0179] AAV8-empty (Lot 19-564E)/AAV8-CMV-GFP (Lot 18-737) in 1×PBS buffer containing 0.001% (w/v) Pluronic F-68, and 0.22 μm filter sterilized.

[0180] AAV2-empty (Lot 19-604E)/AAV2-CMV-GFP (Lot 17-600) in 1×PBS buffer containing 0.001% (w/v) Pluronic F-68, 100 mM sodium citrate and 0.22 μm filter sterilized.

[0181] AAV6-empty (Lot 19-540E)/AAV2-CMV-GFP (Lot 19-718) in 1×PBS buffer containing 0.001% (w/v) Pluronic F-68, 100 mM sodium citrate and 0.22 μm filter sterilized.

[0182] NMR

[0183] Transverse relaxation rates (R.sub.2) or times (T.sub.2) were recorded with a Bruker mini spec mq20 spectrometer (20 MHz; Bruker BioSpin GmbH, Rheinstetten, Germany). The spectrometer was equipped with a 0.47 T magnet and a H2O-10-25AVGX4 probe. At least 4 acquisitions were measured for each sample with sample volumes of 900 μl at 20° C. To determine T.sub.2 or R.sub.2, signal decay was followed for at least 5 sec.

[0184] Preparation of AAV Samples

[0185] The samples were generated by mixing full and empty AAV2, AAV6 and AAV8, respectively, having the same concentration (2*10.sup.13 vg/ml) dissolved in the same aqueous solution comprising 1×PBS buffer containing 0.001% (w/v) Pluronic F-68 (for AAV8), and containing 0.001% (w/v) Pluronic F-68 as well as 100 mM sodium citrate (for AAV2 and AAV6) at different ratios. Samples were measured without further longtime storage, e.g. <0° C.

Example 1

[0186] Determination of the Transverse Relaxation Times for Different Ratios of DNA-Containing and Empty AAV Particles

[0187] DNA-containing AAV particles and empty AAV particles were mixed in aqueous solution (AAV2 and AAV6: 1×PBS buffer containing 0.001% (w/v) Pluronic F-68, 100 mM sodium citrate; AAV8 without sodium citrate) to result in different full/empty ratios spanning the range from 0% to 100% DNA-containing AAV particle. This has been done for AAV particles of the serotypes 2, 6 and 8. For the individual samples, the transverse relaxation times (T.sub.2) were recorded as outlined in the Materials and Methods section above. The results are presented in the following Tables.

TABLE-US-00001 TABLE 1 T.sub.2 values for differently DNA-containing AAV2 particle samples. concentration fraction transverse relaxation time [ms] ratio of DNA-containing standard full/empty AAV2 particles experiment 1 experiment 2 average deviation  0:100 0 vp/mL 0.00 1559.9 1566.9 1563.4 3.5 25:75 0.5*10.sup.13 vp/mL 0.25 1605.8 1568.0 1586.9 18.9 50:50 1*10.sup.13 vp/mL 0.5 1627.0 1602.4 1614.7 12.3 75:25 1.5* 10.sup.13 vp/mL 0.75 1677.6 1662.4 1670.0 7.6 100:0  2*10.sup.13 vp/mL 1.00 1750.6 1770.3 1760.5 9.9

TABLE-US-00002 TABLE 2 T.sub.2 values for differently DNA-containing AAV6 particle samples. concentration fraction transverse relaxation time [ms] ratio of DNA-containing standard full/empty AAV6 particles experiment 1 experiment 2 average deviation  0:100 0 vp/mL 0.00 1629.9 1614.2 1622.1 7.9 25:75 0.5*10.sup.13 vp/mL 0.25 1664.6 1629.1 1646.9 17.8 50:50 1*10.sup.13 vp/mL 0.5 1700.7 1673.8 1687.3 13.5 75:25 1.5* 10.sup.13 vp/mL 0.75 1688.5 1688.5 1688.5 0.0 100:0  2*10.sup.13 vp/mL 1.00 1757.5 1786.1 1771.8 14.3

[0188] The value for the 75:25 ratio was excluded for the fitting calculation due to being deemed to be an experimental error.

TABLE-US-00003 TABLE 3 T.sub.2 values for differently DNA-containing AAV8 particle samples. ratio concentration fraction transverse full/empty of DNA-containing AAV8 particles relaxation time [ms]  0:100 0 vp/mL 0.00 1704.2 25:75 0.5*10.sup.13 vp/mL 0.25 1668.0 50:50 1*10.sup.13 vp/mL 0.5 1635.6 75:25 1.5*10.sup.13 vp/mL 0.75 1608.9 100:0  2*10.sup.13 vp/mL 1.00 1497.3

[0189] The obtained transverse relaxation times were fitted using linear and second order polynomial functions. The results are presented in the following Table 4.

TABLE-US-00004 TABLE 4 Fitting results. For the serotype 6 the fitting is shown including (in brackets) and excluding the data for the 75:25 ratio. AAV linear fitting serotype function R.sup.2 value 2 1.9088*x + 1543.7 0.9208 6 1.5298*x + 1615.1 0.9905 (1.3646*x + 1615.1) (0.8984) 8 −1.8916*x + 1717.4  0.9026 AAV 2.sup.nd order polynomial fitting serotype function R.sup.2 value 2 0.0184*x.sup.2 + 0.0642*x + 1566.7 0.996  6 0.0047*x.sup.2 + 1.0479*x + 1620.9 0.9987 (0.0089*x.sup.2 + 0.4749*x + 1626.2) (0.9318) 8 −0.0166*x.sup.2 − 0.2333*x + 1696.7  0.9633