Genome integrity analysis of adeno-associated virus vectors
A CE based method and kit for the determination of the size and purity of an AAV genome which relies on Capillary Electrophoresis-Laser Induced Fluorescence (CE-LIF) analysis. These methods and kits are capable of detecting intact and partial genomes in a virus vectors such as adeno-associated viruses as well as remove small size impurities. In one example, the method can include creating a nucleic acid ladder using CE-LIF, releasing the genome from within an adeno-associated virus, purifying said genome and analyzing said genome using CE-LIF and comparing the results of the analysis of the genome to the nucleic acid ladder to determine a size of nucleic acids in the genome.
1 . A kit for analyzing an adeno-associated virus comprising:
a standard comprising a plurality of different ssRNA strands, each of the different ssRNA strands having a known and different size;
a positive control containing an intact AAV genome, a partial AAV genome and small size impurities; and
a separation matrix adapted for use with capillary electrophoresis.
2 . The kit of claim 1 further comprising enzymes to digest nucleic acids.
3 . The kit of claim 2 wherein the enzymes comprises benzonase.
4 . The kit of claim 1 further comprising an enzyme that releases nucleic acid from an adeno-associated virus.
5 . The kit of claim 4 wherein said enzyme is Proteinase K.
6 . The kit of claim 1 further comprising materials adapted to perform a PCR purification.
7 . A method for analyzing a genome of an adeno-associated virus in a sample comprising:
creating a standard comprising a number of different nucleic acid strands, each of the different nucleic acid strands having a known size, wherein the nucleic acid strands are ssRNA;
separating each of the different nucleic acid strands in the standard by measuring each of the nucleic acid strands having a known size by utilizing a capillary electrophoresis-laser induced fluorescence (CE-LIF) device and measuring the separated nucleic acid strands as intensity peaks that are a function of migration time using a detector of the CE-LIF;
creating a purified sample by subjecting said sample to purification to remove nucleic acid impurities;
releasing nucleic acid material from the adeno-associated virus in the purified sample by use of an enzyme;
separating and analyzing the released nucleic acid material from the purified sample utilizing the CE-LIF device and measuring the separated nucleic acid strands as one or more intensity peaks that are a function of migration time using a detector of the CE-LIF; and
identifying the size of the nucleic acid material in the sample by comparing the intensities and migration times of the released nucleic acid material from the purified sample and the intensities and migration times of the nucleic acid strands of the standard.
8 . The method of claim 7 wherein the nucleic acid strands of the standard comprise RNA.
9 . The method of claim 7 wherein purification of the samples is performed by digesting the sample with benzonase and isolating the adeno-associated virus by filtration.
10 . The method of claim 7 wherein the enzyme is Proteinase K.
11 . The method of claim 7 wherein the released nucleic acid material is subjected to purification using a PCR purification kit.
12 . The method of claim 7 wherein the nucleic acid material comprises DNA.