Detecting and measuring inverted terminal repeat deletions by capillary electrophoresis

Abstract

The present disclosure provides methods for assessing the integrity of inverted terminal repeat sequences in AAV plasmids by restriction digestion followed by capillary electrophoresis.

Description

DETECTING AND MEASURING INVERTED TERMINAL REPEAT DELETIONSBY CAPILLARY ELECTROPHORESISCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to, and the benefit of. United States Provisional Application No. 63 / 651,222, which was filed on May 23, 2024, the disclosure of which is hereby incorporated by reference in its entirety.BACKGROUND

[0002] Adeno-associated virus (AAV) are non-enveloped single stranded DNA viruses, which were originally discovered as a contaminant in adenovirus preparations. AAVs require the use of a helper virus for coinfection in order to replicate, which may result in an immune response in the host. AAV is the most widely used viral vector for in vivo gene therapy applications. AAVs have low immunogenicity and can enable long-term, stable gene expression.

[0003] The AAV viral genome consists of two T-shaped inverted terminal repeats (ITRs) which flank three genes, the Rep (Replication), Cap (Capsid) and aap (Assembly). AAVs are commonly used as vectors for gene delivery, wherein the viral genes are substituted for a gene of interest. AAV production involves transient transfection of cell lines with the ITR- genetic payload, a separate plasmid containing the viral genes, and a third plasmid containing the helper virus genes.

[0004] One of the major challenges for utilizing AAVs is the stability of the ITR sequence. ITR sequences are highly unstable due to their secondary' structure, complex palindrome sequence, and their high guanine-cytosine content. Thus, quality control testing of ITR-containing plasmids is important to the integrity of the plasmid. Traditionally, ITR integrity has been carried out using restriction enzyme digestion followed by agarose gel electrophoresis for a qualitative assessment. In addition, both Sanger sequencing and next generation sequencing have been implemented, however, they are not cost-effective and are very time consuming.

[0005] It will be appreciated that a need exists for new and efficient methods to determine and quantify the presence of a deletion or mutation within the ITR. Furthermore, it will be appreciated that a need exists to increase the stability to improve production efficiency and consistency.SUMMARY

[0006] This disclosure provides methods of determining the presence of a deletion or mutation within an inverted terminal repeat (ITR) sequence of a plasmid, comprising: contacting a sample including the plasmid with a restriction enzyme that cuts within the ITR sequence to generate a digested sample having one or more plasmid fragments; and separating said plasmid fragments using capillary electrophoresis (CE) to determine the presence of a deletion or mutation within the ITR sequence.

[0007] In one aspect, the plasmid lacks a deletion or mutation within the ITR and has two ITR sequences, wherein each ITR sequence contains two recognition sequences and two restriction sites. In a specific aspect, each ITR sequence contains two recognition sequences specific for cleavage by a restriction enzyme.

[0008] In one aspect, the restriction enzyme is Xmal, Smal or TspMI.

[0009] In one aspect, the plasmid without a deletion or mutation w ithin the ITR sequence generates at least four fragments. In a specific aspect, two of the at least four fragments are about 11 nucleotides long. In another specific aspect, two of the at least four fragments are about 3.4 kb and about 2.6 kb.

[0010] In one aspect, the plasmid with a deletion or mutation contains one intact ITR sequence including two recognition sequences, each containing two restriction sites. In a specific aspect, the plasmid with the deletion or mutation can generate two fragments.

[0011] In one aspect, the ITR sequence is a recombinant adeno-associated virus (AAV) ITR sequence.

[0012] In one aspect, the ITR sequences comprises between 130 bp and 145 bp. In a specific aspect, the ITR sequence includes a left ITR sequence and right ITR sequence each comprising 145 nucleotides. In another specific aspect, either the left ITR sequence or right ITR sequence comprise 141 bp each. In yet another specific aspect, either the left ITR sequence or right ITR sequence comprise 130 bp each. In another specific aspect, the right ITR is truncated.

[0013] In one aspect, an origin of replication (ORI) is closer to the left ITR.

[0014] In one aspect, an ORI is closer to the right ITR.

[0015] In one aspect, an ORI comprises pUC, R6K or a variant thereof

[0016] In one aspect, an ORI is reversed.

[0017] In one aspect, the plasmid does not contain an antibiotic resistance gene.

[0018] In one aspect, the plasmid contains an exogenous gene of interest (GOI).

[0019] In one aspect, a CE capillary length of 30 cm to 60 cm. In a specific aspect, the CE capillary length is 30 cm.

[0020] In one aspect, the CE capillar}' temperature is about 15 °C to about 60 °C. In a specific aspect, the CE capillary temperature is about 25 °C.

[0021] In one aspect, the CE injection and separation uses reverse polarity .

[0022] In one aspect, the CE injection voltage is 1 kv to about 15 kv. In a specific aspect, the CE injection voltage is 5kV.

[0023] In one aspect, the CE injection time is from 5 seconds to 20 seconds. In a specific aspect, the CE injection time is 10 seconds.

[0024] In one aspect, the CE separation voltage is 5 kV to 25 kV. In a specific aspect, the CE separation voltage is 6 kV.

[0025] In one aspect, the CE separation time is from 10 min to 45 min. In a specific aspect, the CE separation time is 20 min.

[0026] This disclosure further provides methods for quantifying a deletion or mutation within an inverted terminal repeat (ITR) sequence of a plasmid, comprising: contacting a sample including the plasmid with a restriction enzy me that cuts within the ITR sequence to generate a digested sample having one or more plasmid fragments; and separating said plasmid fragments using capillary' electrophoresis (CE) to quantify' a deletion or mutation within the ITR sequence.

[0027] In one aspect, the plasmid lacks a deletion or mutation within the ITR and has two ITR sequences, wherein each ITR sequence contains two recognition sequences and two restriction sites. In a specific aspect, each ITR sequence contains two recognition sequences specific for cleavage by a restriction enzyme.

[0028] In one aspect, the restriction enzyme is Xmal, Smal or TspMI.

[0029] In one aspect, the plasmid without a deletion or mutation within the ITR sequence generates at least four fragments. In a specific aspect, two of the at least four fragments are about 11 nucleotides long. In another specific aspect, two of the at least four fragments are about 3.4 kb and about 2.6 kb.

[0030] In one aspect, the plasmid with a deletion or mutation contains one intact ITR sequence including two recognition sequences, each containing two restriction sites. In a specific aspect, the plasmid with the deletion or mutation can generate two fragments.

[0031] In one aspect, the ITR sequence is a recombinant adeno-associated virus (AAV)ITR sequence.

[0032] In one aspect, the ITR sequences comprises between 130 bp and 145 bp. In a specific aspect, the ITR sequence includes a left ITR sequence and right ITR sequence each comprising 145 nucleotides. In another specific aspect, either the left ITR sequence or right ITR sequence comprise 141 bp each. In yet another specific aspect, either the left ITR sequence or right ITR sequence comprise 130 bp each. In another specific aspect, the right ITR is truncated.

[0033] In one aspect, an origin of replication (ORI) is closer to the left ITR.

[0034] In one aspect, an ORI is closer to the right ITR.

[0035] In one aspect, an ORI comprises pUC, R6K or a variant thereof

[0036] In one aspect, an ORI is reversed.

[0037] In one aspect, the plasmid does not contain an antibiotic resistance gene.

[0038] In one aspect, the plasmid contains an exogenous gene of interest (GOI).

[0039] In one aspect, a CE capillary length of 30 cm to 60 cm. In a specific aspect, the CE capillary length is 30 cm.

[0040] In one aspect, the CE capillary’ temperature is about 15 °C to about 60 °C. In a specific aspect, the CE capillary temperature is about 25 °C.

[0041] In one aspect, the CE injection and separation uses reverse polarity’.

[0042] In one aspect, the CE injection voltage is 1 kv to about 15 kv. In a specific aspect, the CE injection voltage is 5kV.

[0043] In one aspect, the CE injection time is from 5 seconds to 20 seconds. In a specific aspect, the CE injection time is 10 seconds.

[0044] In one aspect, the CE separation voltage is 5 kV to 25 kV. In a specific aspect, the CE separation voltage is 6 kV.

[0045] In one aspect, the CE separation time is from 10 min to 45 min. In a specific aspect, the CE separation time is 20 min.

[0046] This disclosure further provides methods for determining the presence and quantifying a deletion or mutation within an inverted terminal repeat (ITR) sequence of a plasmid, comprising: contacting a sample including the plasmid with a restriction enzyme that cuts within the ITR sequence to generate a digested sample having one or more plasmid fragments; and separating said plasmid fragments using capillary electrophoresis (CE) to determine the presence and quantify a deletion or mutation within the ITR sequence

[0047] In one aspect, the plasmid lacks a deletion or mutation within the ITR and has two ITR sequences, wherein each ITR sequence contains two recognition sequences and tworestriction sites. In a specific aspect, each ITR sequence contains two recognition sequences specific for cleavage by a restriction enzyme.

[0048] In one aspect, the restriction enzyme is Xmal, Smal or TspMI.

[0049] In one aspect, the plasmid without a deletion or mutation within the ITR sequence generates at least four fragments. In a specific aspect, two of the at least four fragments are about 11 nucleotides long. In another specific aspect, two of the at least four fragments are about 3.4 kb and about 2.6 kb.

[0050] In one aspect, the plasmid with a deletion or mutation contains one intact ITR sequence including two recognition sequences, each containing two restriction sites. In a specific aspect, the plasmid with the deletion or mutation can generate two fragments.

[0051] In one aspect, the ITR sequence is a recombinant adeno-associated virus (AAV) ITR sequence.

[0052] In one aspect, the ITR sequences comprises between 130 bp and 145 bp. In a specific aspect, the ITR sequence includes a left ITR sequence and right ITR sequence each comprising 145 nucleotides. In another specific aspect, either the left ITR sequence or right ITR sequence comprise 141 bp each. In yet another specific aspect, either the left ITR sequence or right ITR sequence comprise 130 bp each. In another specific aspect, the right ITR is truncated.

[0053] In one aspect, an origin of replication (ORI) is closer to the left ITR.

[0054] In one aspect, an ORI is closer to the right ITR.

[0055] In one aspect, an ORI comprises pUC, R6K or a variant thereof

[0056] In one aspect, an ORI is reversed.

[0057] In one aspect, the plasmid does not contain an antibiotic resistance gene.

[0058] In one aspect, the plasmid contains an exogenous gene of interest (GOI).

[0059] In one aspect, a CE capillary length of 30 cm to 60 cm. In a specific aspect, the CE capillary length is 30 cm.

[0060] In one aspect, the CE capillary temperature is about 15 °C to about 60 °C. In a specific aspect, the CE capillary temperature is about 25 °C.

[0061] In one aspect, the CE injection and separation uses reverse polarity.

[0062] In one aspect, the CE injection voltage is 1 kv to about 15 kv. In a specific aspect, the CE injection voltage is 5kV.

[0063] In one aspect, the CE injection time is from 5 seconds to 20 seconds. In a specific aspect, the CE injection time is 10 seconds.

[0064] In one aspect, the CE separation voltage is 5 kV to 25 kV. In a specific aspect, the CE separation voltage is 6 kV.

[0065] In one aspect, the CE separation time is from 10 min to 45 min. In a specific aspect, the CE separation time is 20 min.

[0066] This disclosure further provides methods for increasing the stability' of an inverted terminal repeat (ITR) sequence of a plasmid, comprising: shortening the length of at least one of the ITR sequences by at least four basepairs.

[0067] In one aspect, the plasmid lacks a deletion or mutation within the ITR and has two ITR sequences, wherein each ITR sequence contains two recognition sequences and two restriction sites.

[0068] In one aspect, each ITR sequence contains two recognition sequences specific for cleavage by a restriction enzyme. In a specific aspect, the restriction enzyme is Xmal or Smal.

[0069] In one aspect, the plasmid without a deletion or mutation within the ITR sequence generates at least four fragments when contacted by a restriction enzyme. In a specific aspect, two of the at least four fragments are about 11 nucleotides long. In another specific aspect, two of the at least four fragments are about 3.4 kb and about 2.6 kb.

[0070] In one aspect, the plasmid with a deletion or mutation contains one intact ITR sequence including two recognition sequences, each containing two restriction sites. In a specific aspect, the plasmid with the deletion or mutation can generate two fragments when contacted by a restriction enzyme.

[0071] In one aspect, the ITR sequence is a recombinant adeno-associated virus (AAV) ITR sequence.

[0072] In one aspect, the ITR sequences comprises between 130 bp and 145 bp. In a specific aspect, the left ITR sequence or right ITR sequence comprise 141 bp each. In another specific aspect, either the left ITR sequence or right ITR sequence comprise 130 bp each. In yet another specific aspect, the right ITR sequence is truncated.

[0073] In one aspect, an origin of replication (ORI) is closer to the left ITR.

[0074] In one aspect, an ORI is closer to the right ITR.

[0075] In one aspect, an ORI comprises pUC, R6K or a variant thereof.

[0076] In one aspect, an ORI is reversed.

[0077] In one aspect, the plasmid does not contain an antibiotic resistance gene.

[0078] In one aspect, the plasmid contains an exogenous gene of interest (GOI).

[0079] In one aspect, the plasmid is digested by a restriction enzy me to generate fragments of the plasmid.

[0080] In one aspect, the fragments of the plasmid are separated using capillary electrophoresis (CE).

[0081] In one aspect, a CE capillary length of 30 cm to 60 cm. In a specific aspect, the CE capillary length is 30 cm.

[0082] In one aspect, the CE capillary temperature is about 15 °C to about 60 °C. In a specific aspect, the CE capillary temperature is about 25 °C.

[0083] In one aspect, the CE injection and separation uses reverse polarity7.

[0084] In one aspect, the CE injection voltage is 1 kv to about 15 kv. In a specific aspect, the CE injection voltage is 5kV.

[0085] In one aspect, the CE injection time is from 5 seconds to 20 seconds. In a specific aspect, the CE injection time is 10 seconds.

[0086] In one aspect, the CE separation voltage is 5 kV to 25 kV. In a specific aspect, the CE separation voltage is 6 kV.

[0087] In one aspect, the CE separation time is from 10 min to 45 min. In a specific aspect, the CE separation time is 20 min.

[0088] This disclosure also provides methods for determining the stability7of an inverted terminal repeat (ITR) sequence of a plasmid, comprising: shortening the length of at least one of the ITR sequences; contacting a sample including the plasmid with a restriction enzyme that cuts within the ITR sequence to generate a digested sample having one or more plasmid fragments; and separating said plasmid fragments using capillary electrophoresis (CE) to determine the stability of the ITR sequence.

[0089] In one aspect, the plasmid lacks a deletion or mutation within the ITR and has two ITR sequences, wherein each ITR sequence contains two recognition sequences and two restriction sites. In a specific aspect, each ITR sequence contains two recognition sequences specific for cleavage by a restriction enzyme.

[0090] In one aspect, the restriction enzyme is Xmal, Smal or TspMI.

[0091] In one aspect, the plasmid without a deletion or mutation within the ITR sequence generates at least four fragments. In a specific aspect, two of the at least four fragments are about 11 nucleotides long. In another specific aspect, two of the at least four fragments are about 3.4 kb and about 2.6 kb.

[0092] In one aspect, the plasmid with a deletion or mutation contains one intact ITR sequence including two recognition sequences, each containing two restriction sites. In a specific aspect, the plasmid with the deletion or mutation can generate two fragments.

[0093] In one aspect, the ITR sequence is a recombinant adeno-associated virus (AAV) ITR sequence.

[0094] In one aspect, the ITR sequences comprises between 130 bp and 145 bp. In a specific aspect, the ITR sequence includes a left ITR sequence and right ITR sequence each comprising 145 nucleotides. In another specific aspect, either the left ITR sequence or right ITR sequence comprise 141 bp each. In yet another specific aspect, either the left ITR sequence or right ITR sequence comprise 130 bp each. In another specific aspect, the right ITR is truncated.

[0095] In one aspect, an origin of replication (ORI) is closer to the left ITR.

[0096] In one aspect, an ORI is closer to the right ITR.

[0097] In one aspect, an ORI comprises pUC, R6K or a variant thereof

[0098] In one aspect, an ORI is reversed.

[0099] In one aspect, the plasmid does not contain an antibiotic resistance gene.

[0100] In one aspect, the plasmid contains an exogenous gene of interest (GOI).

[0101] In one aspect, a CE capillary length of 30 cm to 60 cm. In a specific aspect, the CE capillary length is 30 cm.

[0102] In one aspect, the CE capillary temperature is about 15 °C to about 60 °C. In a specific aspect, the CE capillary temperature is about 25 °C.

[0103] In one aspect, the CE injection and separation uses reverse polarity.

[0104] In one aspect, the CE injection voltage is 1 kv to about 15 kv. In a specific aspect, the CE injection voltage is 5kV.

[0105] In one aspect, the CE injection time is from 5 seconds to 20 seconds. In a specific aspect, the CE injection time is 10 seconds.

[0106] In one aspect, the CE separation voltage is 5 kV to 25 kV. In a specific aspect, the CE separation voltage is 6 kV.

[0107] In one aspect, the CE separation time is from 10 min to 45 min. In a specific aspect, the CE separation time is 20 min.

[0108] The present disclosure provides a method for increasing the stability of an inverted terminal repeat (ITR) sequence of a plasmid, the method comprising preparing a plasmid comprising an ITR sequence having a length of about 130 basepairs, wherein thestability- is increased as compared to a plasmid comprising an ITR sequence having a length of at least about 145 basepairs.

[0109] The present disclosure provides a method for determining the presence of a deletion or mutation within an inverted terminal repeat (ITR) sequence of a plasmid, the method comprising contacting a sample including the plasmid with a restriction enzyme that cuts within the ITR sequence to generate a digested sample having one or more plasmid fragments, and determining the presence of a deletion or mutation within the ITR sequence by separating the plasmid fragments using capillary electrophoresis (CE) and analyzing the number of fragments, wherein a plasmid without a deletion or mutation within the ITR sequence generates more fragments as compared to a plasmid with a deletion or mutation within the ITR sequence.

[0110] These, and other, aspects of the present disclosure will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. The following description, while indicating various embodiments and numerous specific details thereof, is given by way of illustration and not of limitation. Many substitutions, modifications, additions, or rearrangements may be made within the scope of the present disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0111] The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosed methods, there are shown in the drawing’s exemplary embodiments of the methods; however, the methods are not limited to the specific embodiments disclosed. In the drawings:

[0112] FIG. 1 shows the inverted terminal repeats present in the wild-type AAV, according to an exemplary embodiment.

[0113] FIG. 2A shows plasmid 1 containing two inverted terminal repeats (ITRs) on either side of the gene of interest, according to an exemplary embodiment. The inset depicts the complex palindrome of the ITR, composed of an A, A’, and D with smaller palindromic sequences B, B’, C, C’. The digestion sites are noted in the C and C’ regions.

[0114] FIG. 2B shows agarose gel electrophoresis of the linear isoform and the two expected fragments of the fully digested plasmid, according to an exemplary embodiment.

[0115] FIG. 3A shows the electropherogram of a digested plasmid using Xmal showing the expected fragments at peaks 3 and 4 and the linear isoform at peak 5, according to an exemplary embodiment.

[0116] FIG. 3B shows comparison of the peak area percentage for the five observed peaks from FIG. 3A, according to an exemplary embodiment.

[0117] FIG. 4A shows the comparison of percent linear plasmid isoform obtained after capillary electrophoresis between two lots, according to an exemplary embodiment.

[0118] FIG. 4B shows the quantification of variants observed comparing two different sequencing methods and capillary electrophoresis, according to an exemplary' embodiment.

[0119] FIG. 5A shows the plasmid map for two plasmids varying the proximity and orientation of the origin of replication (ORI) relative to the ITR, according to an exemplary embodiment. For plasmid 1, with the ORI is closest to the left ITR and for plasmid 2 the ORI is closest to the right ITR.

[0120] FIG. 5B shows the comparison of percent linear plasmid isoforms after digestion of different plasmid constructs containing either full-length ITR (145 basepairs (bp)) versus shorter ITRs. according to an exemplary embodiment.

[0121] FIG. 6 shows two plasmid constructs generated with shorter ITR lengths of 141 bp (Plasmid 3) and 130 bp (Plasmid 4), as well as a plasmid containing a reversed origin of replication orientation (Plasmid 5), according to an exemplary embodiment.

[0122] FIG. 7 shows a comparison of plasmids containing three ITR lengths transformed into various E. coli strains and various growth conditions, according to an exemplary’ embodiment.

[0123] FIG. 8 A shows a schematic of three different right ITR region lengths, 145 bp,141 bp. and 130 bp, according to an exemplary embodiment.

[0124] FIG. 8B shows a schematic of a plasmid comprising a 145 bp right ITR region, according to an exemplary' embodiment.

[0125] FIG. 8C shows a comparison of the percentage of hairpin deletion rate in the plasmid versus the hairpin deletion rate in the virus for the full length ITRs, according to an exemplary embodiment.

[0126] FIG. 8D shows a comparison of the percentage of hairpin deletion rate in the plasmid versus the hairpin deletion rate in the virus for the shorter ITRs, according to an exemplary embodiment.

[0127] FIG. 9A shows the comparison of percent linear plasmid isoforms after digestion of the full length ITR (145 bp) with the ORI in standard orientation compared to a full lengthITR (145 bp) with the ORI reversed and further in proximity to the L-ITR, according to an exemplary embodiment.

[0128] FIG. 9B shows the comparison of percent linear plasmid isoforms after digestion of the full length ITR (145 bp) with the ORI in standard orientation compared to a full length ITR (145 bp) with the ORI reversed and further in proximity to the L-ITR, according to an exemplary embodiment.

[0129] FIG. 10A shows a comparison of two plasmid constructs, a typical plasmid (Plasmid 1) and a nanoplasmid (Plasmid 6), according to an exemplary embodiment.

[0130] FIG. 10B shows the comparison of percent linear plasmid isoforms of two different lots of plasmid 1 and the nanoplasmid, according to an exemplary embodiment.

[0131] FIG. 11 shows a comparison of three plasmid maps with a reversed ORI and full length ITR (145 bp)(Plasmid 7), 141 bp ITR (Plasmid 8), and 130 bp ITR (Plasmid 9), according to an exemplar}7embodiment.

[0132] FIG. 12A shows the identification of various variants (mutations and deletions) observed for the full length compared to the truncated ITR using long read sequencing, according to an exemplary embodiment.

[0133] FIG. 12B shows the identification of hairpin deletions observed for the full length compared to the truncated ITR using long read sequencing, according to an exemplary7embodiment.

[0134] FIG 12C shows the percent ITR deletions observed for the full length compared to the truncated ITR using capillary electrophoresis, according to an exemplary7embodiment.

[0135] FIG. 13A shows the cell culture performance as measured by VCD (E6 / ml) across the plasmid variants, according to an exemplary7embodiment. The error bars shown represent one standard deviation from the mean.

[0136] FIG. 13B shows the cell culture performance as measured by Glue (g / L) across the plasmid variants, according to an exemplary7embodiment. The error bars shown represent one standard deviation from the mean.

[0137] FIG. 13C shows the cell culture performance as measured by viability (%) across the plasmid variants, according to an exemplary embodiment. The error bars shown represent one standard deviation from the mean.

[0138] FIG. 13D shows the cell culture performance as measured by Glu (mM) across the plasmid variants, according to an exemplary embodiment. The error bars shown represent one standard deviation from the mean.

[0139] FIG. 13E shows the cell culture performance as measured by Lac (g / L) across the plasmid variants, according to an exemplary embodiment. The error bars shown represent one standard deviation from the mean.

[0140] FIG. 13F shows the cell culture performance as measured by NH4 (mM) across the plasmid variants, according to an exemplary embodiment. The error bars shown represent one standard deviation from the mean.

[0141] FIG. 14 shows the comparability of quality and productivity attributes for the plasmid variants, according to an exemplary embodiment. The error bars represent one standard deviation from the mean - * is p<0.05; ** is p<0.01; ns is “not significant”.

[0142] FIG. 15 compares the mRNA expression in the liver of mice for the 145 bp, 141 bp, and 130bp ITRs, according to an exemplary embodiment.

[0143] FIG. 16 shows the plasmid constructs utilized to compare the amount of ITR deletions, according to an exemplary' embodiment.

[0144] FIG. 17 compares the percentage of ITR deletion calculated using capillary' electrophoresis across several different lots, including the nanoplasmid, according to an exemplary embodiment.

[0145] FIG. 18A shows the electropherogram for lot A (#1), according to an exemplary embodiment.

[0146] FIG. 18B shows the percent area calculated for the three peaks observed for lot A (#1), according to an exemplary embodiment.

[0147] FIG. 19A shows the electropherogram for lot B (#2), according to an exemplary embodiment.

[0148] FIG. 19B shows the percent area calculated for the three peaks observed for lot B (#2), according to an exemplary embodiment.

[0149] FIG. 20A shows the electropherogram for lot C (#3), according to an exemplary embodiment.

[0150] FIG. 20B shows the percent area calculated for the three peaks observed for lot C (#3), according to an exemplary embodiment.

[0151] FIG. 21 A shows the electropherogram for lot D (#4). according to an exemplary embodiment.

[0152] FIG. 21B shows the percent area calculated for the three peaks observed for lot D (#4), according to an exemplary embodiment.

[0153] FIG. 22A shows the electropherogram for lot E (#5), according to an exemplary embodiment.

[0154] FIG. 22B shows the percent area calculated for the three peaks observed for lot E (#5), according to an exemplary embodiment.

[0155] FIG. 23A shows the electropherogram for lot F (#6), according to an exemplary embodiment.

[0156] FIG. 23B shows the percent area calculated for the three peaks observed for lot F (#6), according to an exemplary embodiment.

[0157] FIG. 24A shows the electropherogram for lot G (#7). according to an exemplary embodiment.

[0158] FIG. 24B shows the percent area calculated for the three peaks observed for lot F (#7), according to an exemplary embodiment

[0159] FIG. 25A shows the electropherogram for lot H (#8), according to an exemplary embodiment.

[0160] FIG. 25B shows the percent area calculated for the three peaks observed for lot H (#8), according to an exemplary embodiment.

[0161] FIG. 26A shows the electropherogram for lot I (#9), according to an exemplary’ embodiment.

[0162] FIG. 26B shows the percent area calculated for the three peaks observed for lot I (#9), according to an exemplary embodiment.

[0163] FIG. 27 A shows the electropherogram for lot J (#10), according to an exemplary embodiment.

[0164] FIG. 27B shows the percent area calculated for the three peaks observed for lot J (#10), according to an exemplary’ embodiment.

[0165] FIG. 28A shows the electropherogram for lot K (#11), according to an exemplary' embodiment.

[0166] FIG. 28B shows the percent area calculated for the three peaks observed for lot K (#11), according to an exemplary embodiment.

[0167] FIG. 29 A shows the electropherogram for lot L (#12), according to an exemplary' embodiment.

[0168] FIG. 29B shows the percent area calculated for the three peaks observed for lot L (#12), according to an exemplary embodiment.

[0169] FIG. 30A shows the electropherogram for lot M (#13), according to an exemplary embodiment.

[0170] FIG. 30B shows the percent area calculated for the three peaks observed for lot M (#13), according to an exemplary embodiment.

[0171] FIG. 31A shows the electropherogram for lot N (#14), according to an exemplary' embodiment.

[0172] FIG. 3 IB shows the percent area calculated for the three peaks observed for lot N (#14), according to an exemplary embodiment.

[0173] FIG. 32A shows the electropherogram for lot O (#15), according to an exemplary7embodiment.

[0174] FIG. 32B shows the percent area calculated for the three peaks observed for lot O (#15), according to an exemplary embodiment.

[0175] FIG. 33A shows the electropherogram for lot P (#16), according to an exemplary' embodiment.

[0176] FIG. 33B shows the percent area calculated for the three peaks observed for lot P (#16), according to an exemplary embodiment.

[0177] FIG. 34A shows the electropherogram for lot Q (#17), according to an exemplary embodiment.

[0178] FIG. 34B shows the percent area calculated for the three peaks observed for lot Q (#17), according to an exemplary embodiment.

[0179] FIG. 35 A shows the electropherogram for lot R (#18), according to an exemplary embodiment.

[0180] FIG. 35B shows the percent area calculated for the three peaks observed for lot R (#18), according to an exemplary embodiment.

[0181] FIG. 36A shows the electropherogram for lot S (#19), according to an exemplary' embodiment.

[0182] FIG. 36B shows the percent area calculated for the three peaks observed for lot S (#19), according to an exemplary embodiment.

[0183] FIG. 37 compares the percentage of ITR deletions determined across several lots, according to an exemplary' embodiment.DETAILED DESCRIPTION

[0184] The disclosed methods may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure. It is to be understood that the disclosed methods are not limited to the specific methods described and / or shown herein, and that the terminology' used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed methods.

[0185] Unless specifically stated otherwise, any description as to a possible mechanism or mode of action or reason for improvement is meant to be illustrative only, and the disclosed methods are not to be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement.

[0186] Where a range of numerical values is recited or established herein, the range includes the endpoints thereof and all the individual integers and fractions within the range, and also includes each of the narrower ranges therein formed by all the various possible combinations of those endpoints and internal integers and fractions to form subgroups of the larger group of values within the stated range to the same extent as if each of those narrower ranges was explicitly recited. Where a range of numerical values is stated herein as being greater than a stated value, the range is nevertheless finite and is bounded on its upper end by a value that is operable within the context of the herein disclosure. Where a range of numerical values is stated herein as being less than a stated value, the range is nevertheless bounded on its lower end by a non-zero value. It is not intended that the scope of the methods be limited to the specific values recited when defining a range. All ranges are inclusive and combinable.

[0187] When values are expressed as approximations, by use of the antecedent "about." it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise.

[0188] It is to be appreciated that certain features of the disclosed methods which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed methods that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.

[0189] In order to manufacture biopharmaceutical products for gene therapy, it is important to manufacture a stable product. Adeno-associated viruses (AAVs) have been widely used as gene delivery vectors to deliver genetic material, such as delivering nucleic acids for gene therapy. AAVs provide the advantages of non-pathogenicity and low immunogenicity. AAVs are nonpathogenic members of the Parvoviridae family under Dependovirus genus and require helpers, such as Adenovirus or Herpesvirus, for infection (Venkatakrishnan et al., Structure and Dynamics of Adeno-Associated Virus Serotype 1 VP1- Unique N-Terminal Domain and Its Role in Capsid Trafficking, Journal of Virology. May, 2013, vol. 87, no. 9, pages 4974-4984). AAV encapsulates a single-stranded DNAgenome of about 4.8 kilobases (kb) in an icosahedral capsid which is made of a shell of capsid viral proteins. Recombinant AAV genomes are nonpathogenic and do not integrate into a host’s genome, but exist as stable episomes that provide long-term expression. AAV serotypes make a very useful system for preferentially transducing specific cell types.

[0190] Overall, AAV-based therapy has the advantages of being non-pathogenic and non-toxic, having cell type-specific infection, and offering different serotypes with varying cell transduction efficiencies. A disadvantage is that AAV production, purification, and characterization are more complex compared to, for example, antibody therapies. Fully packaged AAVs consist of an icosahedral capsid containing an about 4.8 kb single-stranded genome. An empty capsid has a molecular weight of about 3750 kDa, while a full capsid with an about 4.7 kb single-stranded genome has a molecular weight of about 5100 kDa. The purity of AAVs is defined by several product-related impurities, including empty capsids, capsids containing partial or incorrect genomes, and aggregated or degraded capsid, as well as residual HCPs.

[0191] One unique feature of AAVs is the presence of inverted terminal repeats (ITR) located at either end of the single-stranded DNA molecule. ITRs play critical roles in the replication, assembly, packaging and delivery to the host genome. Each ITR consists of a complex palindromic sequence composed of smaller palindromic sequences. The D region is 21 basepairs in length, is the only non-palindrome sequence present in the ITR and is necessary for packaging. The larger A and A’ segments are about 40 bp each and contain the Rep binding elements as well as the Terminal resolution sites and form the stem of the molecule. The B, B’, C and C’ regions are composed of nine basepairs each and comprise the hairpin structure at the end of molecule, and contains several restriction enzyme sequences. The B and B’ segments are mostly adenine and thymine rich and form one loop, while the C and C’ segments are mostly guanine and cytosine (G-C) rich and form the second loop. The arrangement of the B,B’ and C,C’ palindromic sequences determines the orientation of the ITR. In the “flip” orientation, the B,B’ palindrome sequence is closer to the 3’ end of the ITR, whereas the C,C’ palindrome sequence is closer to the 3’ end of the ITR in the “flop” orientation.

[0192] Due to the high G-C rich content, their palindromic sequence and the hairpin structure, ITRs are quite unstable. This instability causes mutations and deletions to occur, which could possibly affect production of the AAV. Thus, the characterization of these mutations and / or deletions is important to understanding ITR integrity. Several methods have been implemented for quality' control of AAV-ITR containing plasmids. Traditionally,ITR integrity has been carried out using restriction enzyme digestion followed by agarose gel electrophoresis for a qualitative assessment. In addition, both sanger sequencing and next generation sequencing have been implemented, however they are not cost-effective and are very time consuming. Furthermore, Sanger sequencing cannot reliably sequence through the difficult ITR sequence. Capillary Electrophoresis (CE) can perform size separation and quantitation using the peak area in the calculation, however it does not reveal the nature of the mutation as it does not provide any sequence information. Next Generation sequencing provides greater depth than Sanger sequencing and can quantify the variants present in the sample and identify major and minor variants.

[0193] The present application provides methods for determining the presence of a deletion or mutation within an inverted terminal repeat (ITR) sequence of a plasmid. In some exemplary embodiments, the method provides an efficient and cost-effective approach to detecting a deletion or mutation within an ITR sequence. This approach can be applied to other plasmid-based products and can be used to identify potential variants which may affect the quality of the products.

[0194] This disclosure provides methods to satisfy the aforementioned demands by providing methods to determine the presence of a deletion or mutation within an ITR. Exemplary' embodiments disclosed herein satisfy the aforementioned demands and the long- felt needs.

[0195] The present disclosure provides a method for determining the presence of a deletion or mutation within an inverted terminal repeat (ITR) sequence of a plasmid, comprising contacting a sample including the plasmid with a restriction enzy me that cleaves within the ITR sequence to generate a digested sample having one or more plasmid fragments, and determining the presence of a deletion or mutation within the ITR sequence by separating the plasmid fragments using capillary electrophoresis (CE) and analyzing the number of fragments, wherein a plasmid without a deletion or mutation within the ITR sequence generates more fragments as compared to a plasmid with a deletion or mutation within the ITR sequence.

[0196] The presence of a deletion or mutation within an inverted terminal repeat (ITR) sequence of a plasmid may be quantified by measuring the relative abundance of each fragment peak separated by capillary' electrophoresis.

[0197] The presence of a deletion or mutation within an inverted terminal repeat (ITR) sequence of a plasmid may be quantified by measuring the area of each fragment peak separated by capillary electrophoresis.

[0198] The presence of a deletion or mutation within an inverted terminal repeat (ITR) sequence of a plasmid may be determined by the number of fragment peaks separated by capillary electrophoresis.

[0199] The ITR sequence may contain two restriction enzy me sites. In a plasmid comprising two ITR sequences, the plasmid may contain four restriction enzy me sites.

[0200] The restriction enzyme may be Xmal. Smal or TspMI.

[0201] In a plasmid comprising two ITR sequences and four restriction enzyme sites, cleavage by the restriction enzyme may generate at least four fragments. Two of the at least four fragments may be about 11 nucleotides long. Two of the at least four fragments may be about 2.6 kb to about 3.4 kb long.

[0202] Without wishing to be bound by theory, a plasmid comprising two ITR sequences comprising four restriction enzyme sites that do not contain a deletion or mutation will generate two short (about 11 nucleotides) and two long (about 2.6 kb to about 3.4 kb) fragments, while a plasmid comprising two ITR sequences comprising four restriction enzyme sites that contain a deletion or mutation will generate less than four fragments because one or more of the restriction enzyme sites will fail to be cleaved.

[0203] A plasmid with increased stability7may lack a deletion or mutation within the ITR sequence.

[0204] A plasmid with an ITR shorter than 145 bp will be more stable across various E. coli strains and growth conditions.

[0205] Without wishing to be bound by theory, reversing the orientation of the origin of replication may improve plasmid stability. Without wishing to be bound by theory7, an origin of replication about 400 to about 600 basepairs away from the ITR sequence may reduce mutation rate of the plasmid.

[0206] The ITR sequence may be a recombinant adeno-associated virus (AAV) ITR sequence. The AAV ITR sequence may be an AAV2 serotype ITR sequence.

[0207] The inventors of the present disclosure surprisingly found that plasmid fragments following cleavage by the restriction enzyme may be analyzed quantitatively using the capillary electrophoresis (CE) methods disclosed herein. Because the analyzed fragments were double stranded DNA derived from enzy matic cleavage of a plasmid and not single stranded DNA, the sample did not need to be denatured.

[0208] The CE capillary7length may be about 30 cm to about 60 cm. The CE capillary length may be about 30 cm.

[0209] The CE capillary' temperature may be about 15 °C to about 60 °C. The CE capillary temperature may be about 25 °C.

[0210] The CE injection and separation may use reverse polarity.

[0211] The CE injection voltage may be about 1 kV to about 30 kV. The CE injection voltage may be about 5kV.

[0212] The CE injection time may be from about 5 seconds to about 20 seconds. The CE injection time may be about 10 seconds.

[0213] The CE separation voltage may be from about 5 kV to about 25 kV. The CE separation voltage may be about 6 kV.

[0214] The CE separation time may be from about 10 min to about 45 min. The CE separation time may be about 20 min.

[0215] The present disclosure provides a method for increasing the stability of an inverted terminal repeat (ITR) sequence of a plasmid, comprising preparing a plasmid comprising an ITR sequence having a length of about 130 basepairs, wherein the stability' is increased as compared to a plasmid comprising an ITR sequence having a length of at least about 145 basepairs.

[0216] The ITR sequence may have a length of about 130 basepairs. The ITR sequence may have a length of about 133 basepairs. The ITR sequence may have a length of about 135 basepairs. The ITR sequence may have a length of about 137 basepairs. The ITR sequence may have a length of about 140 basepairs. The ITR sequence may have a length of about 141 basepairs. The ITR sequence may have a length of about 142 basepairs. The ITR sequence may have a length of about 144 basepairs.

[0217] The ITR sequence may contain two restriction enzyme sites.

[0218] The restriction enzyme may be Xmal or Smal.

[0219] The plasmid may comprise an origin of replication (ORI) that is about 400 to about 600 basepairs away from the ITR sequence.

[0220] The plasmid may comprise an ORI that is reverse oriented 3' to 5'. The reverse oriented ORI may be about 70 to about 200 basepairs away from the ITR sequence.

[0221] Unless described otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing, particular methods and materials are now described.

[0222] The term “a” should be understood to mean “at least one'’ and the terms “about’' and “approximately” should be understood to permit standard variation as would be understood by those of ordinary skill in the art, and where ranges are provided, endpoints are included. As used herein, the terms “include,” “includes,” and “including” are meant to be non-limiting and are understood to mean “comprise,” “comprises,” and “comprising” respectively.

[0223] As used herein, a “sample” refers to a mixture of molecules that comprises at least an oligonucleotide, such as a DNA oligonucleotide, that is subjected to manipulation in accordance with the methods of the disclosure, including, for example, separating, analyzing, extracting, concentrating, profiling and the like.

[0224] The terms “nucleic acid”, “oligonucleotide”, “polynucleotide” are used interchangeably. They refer to either naturally-occurring or synthetic polymeric forms of nucleotides. The oligonucleotides of the present disclosure may be ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecules. Nucleic acids are composed of nucleotides which include a sugar, phosphate group and a nitrogenous base. The nucleotides include adenine (A), cytosine (C), guanine (G), thymine (T) and Uracil (U).

[0225] The term “DNA” or “DNA molecule” refers to a string of deoxyribonucleotides. The term “RNA” or “RNA molecule” refers to a string of ribonucleotides. DNA and RNA can be single-stranded or double-stranded. The person skilled in the art will understand the four nucleotide monomers of DNA are A. C, G and T and would further understand the “T” would be replaced with “U” in an RNA sequence.

[0226] A "vector," as used herein, refers to a recombinant plasmid or virus (“viral vector”) that comprises a nucleic acid to be delivered into a host cell, either in vitro or in vivo. Vectors derived from AAV are particularly attractive for delivering genetic material because (i) they are able to infect (transduce) a wide variety of non-dividing and dividing cell types including muscle fibers and neurons; (ii) they are devoid of the virus structural genes, thereby eliminating the natural host cell responses to virus infection, for example, interferon- mediated responses; (iii) wild type AAVs have never been associated with any pathology in humans; (iv) in contrast to wild type AAVs. which are capable of integrating into the host cell genome, replication-deficient AAV vectors generally persist as episomes, thus limiting the risk of insertional mutagenesis or activation of oncogenes; and (v) in contrast to other vector systems, AAV vectors do not trigger a significant immune response (see ii), thus granting long-term expression of the therapeutic transgenes (provided their gene products are not rejected).

[0227] A "recombinant viral vector" refers to a recombinant polynucleotide vector including one or more heterologous sequences (e.g., nucleic acid sequence not of viral origin).

[0228] A "recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector including one or more heterologous sequences (e.g., nucleic acid sequence not of AAV origin) that may be flanked by at least one, e.g., two, AAV inverted terminal repeat sequences (ITRs). Such rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper virus (or that is expressing suitable helper functions) and that is expressing AAV rep and cap gene products (e.g. AAV Rep and Cap proteins).

[0229] The term "plasmid’' refers to a small, circular double stranded DNA molecule. Often a plasmid is a separate DNA molecule from the chromosomal DNA which is capable of replicating independently from the chromosomal DNA. Plasmids typically contain an origin of replication, a multiple cloning site where restriction enzymes will cleave to allow for insertion of a gene of interest, and an antibiotic resistance gene. In some aspects, a plasmid can also be a cosmid, bacterial artificial chromosome (BAC), bacteriophages, viral vectors, or hybrids thereof.

[0230] The term “origin of replication” or “ORI” is a region on the plasmid specified to initiate replication. Non-limiting examples can include pUC or R6K.

[0231] The term “inverted terminal repeat” or “ITR” is a DNA sequence located on either side of the genetic information to be packaged within the AAV molecule.

[0232] The term “gene” refers to a sequence of nucleotides, which may be naturally occurring or synthesized. The gene may be a full sequence of DNA that when synthesized and translated, leads to produce a functional protein. The gene may also be a part of a functional product.

[0233] The term “complementarity” refers to a nucleic acid sequence that forms hydrogen bonds with another nucleic acid sequence.

[0234] The term “repeat region” should be understood to mean a region of a DNA sequence that contains a pattern of nucleotides that occur in several copies in said region. Repeated sequences can consist of two to several thousand nucleotides repeated in tandem.

[0235] The term “sequencing read” or “read” should be understood to mean the DNA sequence of a fragment of DNA produced from a sequencer.

[0236] The term “read length” should be understood to mean the length of a sequencing read in units of nucleotides.

[0237] The term “long read” should be understood to mean sequencing reads in excess of 500 nucleotides in length. Long read data is typically produced via third generation, or Next-Next-Gen, Sequencing platform, and produces an average read length in excess of 10,000 nucleotides (or 10 kb). In contrast, short-read Next-Gen sequencing produces an average read length of 150 nucleotides.

[0238] The term “amplified” should be understood to mean DNA sequences that have been replicated for sequencing using an artificial method. Samples are amplified prior to DNA sequencing to increase the number of target sequences to be sequenced, and thus increase the number of reads present in the sequencing data produced by the sequencer.

[0239] The term “PacBio” should be understood to mean PacBio sequencing, a trademarked sequencing method from Pacific Biosciences.

[0240] A "viral particle" refers to a particle composed of at least one viral capsid protein and an encapsulated viral genome. While AAV is described in this disclosure as a model virus or viral particle, it is contemplated that the disclosed methods can be applied to profile a variety of viruses, e.g.. the viral families, subfamilies, and genera. In some aspects, the viral capsid, virus, or viral particle belongs to a viral family selected from the group consisting of Adenoviridae, Parvoviridae, Retroviridae, Baculoviridae, and Herpesviridae. In some aspects, the viral capsid, virus, or viral particle belongs to a viral genus selected from the group consisting of Atadenovirus, Aviadenovirus. Ichtadenovirus, Mastadenovirus, Siadenovirus, Ambidensovirus, Brevidensovirus, Hepandensovirus, Iteradensovirus, Penstyldensovirus, Amdoparvovirus, Aveparvovirus, Bocaparvovirus, Copiparvovirus, Dependoparvovirus, Erythroparvovirus. Protoparvovirus. Tetraparvovirus, Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, Lentivirus, Spumavirus, Alphabaculovirus, Betabaculovirus, Deltabaculovirus, Gammabaculovirus. Iltovirus. Mardivirus, Simplexvirus, Varicellovirus, Cytomegalovirus, Muromegalovirus, Proboscivirus, Roseolovirus, Lymphocryptovirus, Macavirus, Percavirus, and Rhadinovirus.

[0241] As used herein, the term “gene therapy” is a method of treatment of a genetic disease by modifying or manipulating a gene of interest. The key step in gene therapy is efficient delivery of the vector to the appropriate tissue or cells. Non-limiting examples of gene therapy products can include plasmid DNA, viral vectors, non-viral vectors, bacterial vectors, human gene editing technology, and patient-derived cellular gene therapy products.

[0242] As used herein, the term “digestion” refers to hydrolysis of one or more peptide bonds of a protein. There are several approaches to carrying out digestion of a protein in a sample using an appropriate hydrolyzing agent, for example, enzymatic digestion or non-enzymatic digestion. Digestion of a protein into constituent peptides can produce a "peptide digest” that can further be analyzed using peptide mapping analysis.

[0243] As used herein, the term “digestive enzyme” refers to any of a large number of different agents that can perform digestion of a protein. Non-limiting examples of hydrolyzing agents that can carry' out enzymatic digestion include protease from Aspergillus Saitoi, elastase, subtilisin, protease XIII. pepsin, trypsin, Tryp-N, chymotrypsin, aspergillopepsin I. LysN protease (Lys-N), LysC endoproteinase (Lys-C), endoproteinase Asp-N (Asp-N), endoproteinase Arg-C (Arg-C), endoproteinase Glu-C (Glu-C) or outer membrane protein T (OmpT), immunoglobulin-degrading enzy me of Streptococcus pyogenes (IdeS), thermolysin, papain, pronase, V8 protease or biologically active fragments or homologs thereof or combinations thereof. For a recent review discussing the available techniques for protein digestion see Switazar et al., “Protein Digestion: An Overview of the Available Techniques and Recent Developments” (Linda Switzar. Martin Giera & Wilfried M. A. Niessen, 12 JOURNAL OF PROTEOME RESEARCH 1067-1077 (2013)).

[0244] The term “restriction enzyme” refers to an enzyme that cleaves DNA molecules at or near a specific sequence of bases. A restriction enzyme will cut (or cleave) the double stranded DNA into many pieces, can make a staggered cut, producing ends with an overhang, or by a blunt cut, producing ends with no overhang. Suitable restriction enzy mes include enzymes that cut within the AAV ITR sequences and selection of such enzymes will be within the skill of the person of ordinary skill in the art. Non-limiting examples of restriction enzymes include Smal, Xmal, and EcoRI.

[0245] The term “restriction site” refers to a region of DNA that is between 6-8 nucleotides in length and is the specific location on DNA that a restriction enzy me will cleave. Most restriction sites are palindromic, a double stranded DNA sequence about 3-5 bases in length followed by their complementary bases in reverse order. A non-limiting example includes CCCGGG for Smal.

[0246] The term “isozyme” or “isoenzy me” refers to an enzy me that catalyzes the same reaction however differs in amino acid sequence and molecular shape. This enzyme may have a modified function and may be reactive under different conditions or in different cells or tissues. A non-limiting example of an isozyme is TspMI.

[0247] The term “electrophoresis” refers to an analytical method of separation of molecules in a sample mixture through applying a voltage to the sample. Separation of molecules can be based on size, charge, shape, etc. The matrix upon which separation occurscan be a hydrated gel, such a polyacrylamide gel electrophoresis or through a tube, such as capillary electrophoresis.

[0248] The term “capillary electrophoresis” refers to an analytical method separating ions based on their electrophoretic mobility through a tube, with an applied voltage. Movement of the molecules through the capillary is dependent on their charge and size.

[0249] An alternative embodiment of the present disclosure includes methods of quantifying a deletion or mutation within an inverted terminal repeat (ITR) sequence of a plasmid comprising contacting a sample including the plasmid with a restriction enzyme that cuts within the ITR sequence to generate a digested sample having one or more plasmid fragments; and separating said plasmid fragments using capillary electrophoresis (CE) to quantify a deletion or mutation within the ITR sequence.

[0250] Another alternative embodiment of the present disclosure includes determining the presence and quantifying a deletion or mutation within an inverted terminal repeat (ITR) sequence of a plasmid, comprising contacting a sample including the plasmid with a restriction enzyme that cuts within the ITR sequence to generate a digested sample having one or more plasmid fragments; and separating said plasmid fragments using capillary electrophoresis (CE) to determine the presence and quantify a deletion or mutation within the ITR sequence.

[0251] Another alternative embodiment of the present disclosure includes a method for increasing the stability of an inverted terminal repeat (ITR) sequence of a plasmid, comprising shortening the length of at least one of the ITR sequences by at least four basepairs.

[0252] Another alternative embodiment of the present disclosure includes a method for determining the stability of an inverted terminal repeat (ITR) sequence of a plasmid, comprising: shortening the length of at least one of the ITR sequences; contacting a sample including the plasmid with a restriction enzy me that cuts within the ITR sequence to generate a digested sample having one or more plasmid fragments; and separating said plasmid fragments using capillary’ electrophoresis (CE) to determine the stability of the ITR sequence.

[0253] It is understood that the present disclosure is not limited to any of the aforesaid nucleic acid(s), sample(s), AAV(s), virus(es), serofype(s), plasmid(s), vector(s), digestive enzyme(s), chromatographic method(s), temperature(s), or concentration(s), and any nucleic acid(s). sample(s), AAV(s), virus(es), serotype(s). plasmid(s), vector(s), digestive enzyme(s), chromatographic method(s), temperature(s). or concentration(s) can be selected by any suitable means.

[0254] The present disclosure will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the disclosure.EXAMPLESExample 1. AAV-ITR Integrity

[0255] Plasmids containing inverted terminal repeats (ITR) contain a hairpin structure and 70% GC content which contributes to the instability of these constructs, thus making quality control testing important. Current quality control assays include digestion with restriction enzyme Xmal or Smal followed by separation on agarose gel electrophoresis for separation based on size. However, agarose gel electrophoresis is qualitative in nature, and only indicates the presence of an ITR mutation, but does not indicate the nature of ITR mutations.

[0256] Capillary' electrophoresis (CE) is another method for size separation, which also does not determine the nature of the ITR mutation, however, is a quantitative assay through the use of the peak area. To determine the amount of ITR deletions present for a set of ITR bearing plasmids, a method was conducted with the restriction enzyme Xmal and quantitation using capillary electrophoresis. As shown in FIG. 1, the inverted terminal repeats form a hairpin structure, composed of a complex palindrome, and can either be in a “flip” or “flop” orientation. The overall internal repeat houses two larger palindromic sequences, A and A’, each approximately 40 base pairs each, and four smaller palindromic sequences, B, B’. C and C’, each approximately 9 base pairs each.

[0257] A plasmid was generated which contained two ITR regions, each containing two Smal (or Xmal) restriction enzyme sites, as shown in FIG. 2A. Upon digestion with Xmal followed by agarose gel electrophoresis, three bands were visualized, as shown in FIG. 2B. Two fragments at 2.6 kilobase (kb) and 3.4 kb were observed, and are the expected fragments of the fully digested plasmid. The third band observed on the gel was a 6 kb fragment, which corresponded to the linear isoform of the plasmid, which indicates a mutation of the restriction sites within one of the ITR regions. The digested product was analyzed by capillary electrophoresis, and compared to a double stranded DNA (dsDNA) ladder for size comparison, as shown in FIG. 3A. Five peaks were observed in the electropherogram, and the relative abundance of each peak is shown in FIG. 3B.

[0258] The ability to quantify fragments of a digested plasmid was further tested with a plasmid including a gene of interest (GOI) with 145 bp length w ild-type AAV2 ITRs. Upon digestion of multiple plasmid lots using Xmal, CE analysis revealed varying amounts of thelinear isoform present, as shown in FIG. 4A. A significant increase of the linear isoform was observed when compared to the parental source and lot 001. However, since CE lacks the resolution at the nucleotide level to differentiate between a deletion and a mutation, sequencing assays were performed to further characterize the plasmid. Next-generation sequencing was performed as it provides greater depth than Sanger sequencing, and can quantify the variants present within the sample. Both PacBio long-read sequencing and Illumina short-read sequencing was performed on the digested plasmid sample lot 003. as shown in FIG. 4B. The sequencing data revealed a deletion in the left ITR (L-ITR) causing loss of the Xmal restriction site, and an intact right ITR (R-ITR), resulting in the linear isoform. A BB’ deletion w as also detected; however, it w as not insensitive to Xmal digestion.Example 2. Plasmid design to improve ITR stability

[0259] It was hypothesized that 145 bp ITRs are inherently unstable and prone to mutations. Shorter ITRs are believed to have lower amounts of the linear isoform. The proximity of the origin of replication (ORI) to the ITRs and the orientation of the ORI within the plasmid is hypothesized to also affect ITR stability. Two plasmid maps with varying orientation and proximity of the ORI to the ITRs are shown in FIG. 5A. Upon digestion of several different plasmids w ith Xmal, vary ing amounts of the linear isoform of the plasmid were observed, as shown in FIG. 5B. A significant decrease in the linear plasmid isoform w as observed when comparing a 141 bp ITR and 130 bp ITR to that of the 145 bp ITR length. In addition, an even higher percentage of the linear isoform was observed when the ORI was closest to the R-ITR, suggesting greater instability7of the ITR.

[0260] To evaluate the effect of ITR length and ORI orientation, three constructs were generated, as shown in FIG. 6. Two shorter ITRs were generated, 130 bp (plasmid 4) and 141 bp (plasmid 3), as well as a plasmid (plasmid 5) with an altered backbone containing a reverse ORI (RevOri). The original design had the ORI 420 bp aw ay from the L-ITR, where the RevOri is 95 bp aw ay from the L-ITR. The three new constructs were transformed into various E. coli strains and grow th conditions. As shown in FIG. 7, plasmids with shorter ITRs were more stable across various E. coli strains and growth conditions, when compared to the full length ITR of 145 bp. Interestingly, upon sequencing the transformed plasmids containing the shorter or deleted ITRs, full length ITRs were observed when produced in viruses, as shown in FIG. 8C and FIG. 8D. Fig. 8A and FIG. 8B depict how intramolecular base-pairing between the A’ and truncated A region provides template for replication and repair of the truncated region.

[0261] The effectiveness of reversing the ORI is shown in FIG. 9A and FIG. 9B. In comparing to the full length ITR, the RevOri was significantly more stable as show n by the decrease in the percentage of linear plasmid observed across all E. coli strains and growth conditions.

[0262] In addition to orientation of the ORI, the type of replication origin was evaluated. A nanoplasmid is a compact plasmid, which does not carry any antibiotic resistance, and contains an R6K ORI, as shown in FIG. I0A, plasmid 6. Plasmid 6 was digested with Smal and the percent linear plasmid was evaluated. As shown in FIG. 10B, plasmid 6 has no deletion or mutation within the ITR as evidenced by no linear plasmid detection.Example 3. Manufacturability Assessment of Truncated ITRs

[0263] To evaluate the manufacturability of the ITR deletions, three plasmid constructs w ere generated with a reversed origin of replication and either 145 bp ITR, 141 bp ITR and a 130 bp ITR, as shown in FIG. 11. The plasmids, plasmid 7, plasmid 8 and plasmid 9, were characterized for mutations and deletions in the ITR by both capillary electrophoresis and long read sequencing by PacBio as shown in FIG. 12A, 12B and 12C. Comparable ITR deletions were observed for the truncated ITRs using capillary electrophoresis, and significant decreases were observed in both CC’ and hairpin deletions. Overall, a 3 to 5-fold reduction in ITR variants w ere observed in long-read sequencing.

[0264] The production stage cell culture performance of the three plasmid constructs, plasmid 7, plasmid 8 and plasmid 9. was evaluated. As shown in FIGs. 13A-13F, comparable performance of the three constructs was observed across the conditions tested. In addition, the quality and productivity attributes were comparable at bioreactor harvest, as shown in FIG. 14.Example 4. Case Study of Truncated ITRs

[0265] To evaluate in vivo expression in mice, the three constructs from Example 3 were used. Mice were dosed through IV administration with w ither the 145 bp ITR, the 141 bp ITR or the 130 bp ITR, and expression of mRNA in the liver was examined seven days later. As shown in FIG. 15, mRNA expression of the full length and truncated ITRs express similarly in vivo in mice.Example 5. Case Study of ITR deletions

[0266] To evaluate the lot to lot variability, a set of ITR bearing plasmids w as evaluated using capillary electrophoresis for ITR deletions. Each plasmid (plasmid 10 and plasmid 11) contained the gene of interested with two ITRs. Each ITR contains two Smal restriction sites as shown in FIG. 16. In addition, plasmid 11, a nanoplasmid, was evaluated for ITRdeletions. As shown in FIG. 17, the amount of ITR deletion observed for the various lots of plasmids evaluated was between 0.3% and 4.4%. FIG. 18-35 show the capillary electrophoresis data for each lot evaluated. Plasmid 11 did not show any ITR deletions as evidenced by the detection of two peaks after capillary electrophoresis, as shown in FIG. 36A and 36B. The comparison of the ITR deletions observed using the capillary' electrophoresisbased method is shoyvn in FIG. 37. Based on the results, ITR deletions can consistently be quantified using capillary electrophoresis.

[0267] All references cited herein, including U.S. patent and applications are incorporated by reference in their entirety. The present disclosure is not to be limited in terms to the particular examples described in this application, which are intended as illustrations of various aspects. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims.EMBODIMENTS

[0268] The following list of embodiments is intended to complement, rather than displace or supersede, the previous descriptions.

[0269] Embodiment Set 1:1. A method for determining the presence of a deletion or mutation within an inverted terminal repeat (ITR) sequence of a plasmid, comprising:(a) contacting a sample including the plasmid with a restriction enzyme that cuts within the ITR sequence to generate a digested sample having one or more plasmid fragments; and(b) separating said plasmid fragments using capillary electrophoresis (CE) to determine the presence of a deletion or mutation within the ITR sequence.2. The method of embodiment 1, wherein the plasmid lacks a deletion or mutation within the ITR and has two ITR sequences, wherein each ITR sequence contains two recognition sequences and two restriction sites.3. The method of embodiment 2, wherein each ITR sequence contains two recognition sequences specific for cleavage by a restriction enzyme.4. The method of embodiment 1, wherein the restriction enzy me is Xmal, Smal or TspMI.5. The method of embodiment 2, wherein the plasmid without a deletion or mutation within the ITR sequence generates at least four fragments.6. The method of embodiment 5, wherein two of the at least four fragments are about 11 nucleotides long.7. The method of embodiment 5, wherein two of the at least four fragments are about 3.4 kb and about 2.6 kb.8. The method of embodiment 1. wherein the plasmid with a deletion or mutation contains one intact ITR sequence including two recognition sequences, each containing two restriction sites.9. The method of embodiment 8, wherein the plasmid with the deletion or mutation can generate two fragments.10. The method of embodiment 1, wherein the ITR sequence is a recombinant adeno- associated virus (AAV) ITR sequence.11. The method of embodiment 1, wherein the ITR sequences comprises between 130 bp and 145 bp.12. The method of embodiment 11, wherein the ITR sequence includes a left ITR sequence and right ITR sequence each comprising 145 nucleotides.13. The method of embodiment 11, wherein either the left ITR sequence or right ITR sequence comprise 141 bp each.14. The method of embodiment 11, wherein either the left ITR sequence or right ITR sequence comprise 130 bp each.15. The method of embodiment 11, wherein the right ITR sequence is truncated.1 . The method of embodiment 1, wherein an origin of replication (ORI) is closer to the left ITR.17. The method of embodiment 1, wherein an ORI is closer to the right ITR.18. The method of embodiment 1, wherein an ORI comprises pUC. R6K or a variant thereof.19. The method of embodiment 1 , wherein an ORI is reversed.20. The method of embodiment 1, wherein the plasmid does not contain an antibiotic resistance gene.21. The method of embodiment 1, wherein the plasmid contains an exogenous gene of interest (GOI).22. The method of embodiment 1, wherein a CE capillary length of 30 cm to 60 cm.23. The method of embodiment 22, wherein the CE capillary length is 30 cm.24. The method of embodiment 1, wherein a CE capillary temperature is about 15 °C to about 60 °C.25. The method of embodiment 24, wherein the CE capillary temperature is about 25 °C.26. The method of embodiment 1, wherein a CE injection and separation uses reverse polarity’.27. The method of embodiment 1, wherein a CE injection voltage is 1 kv to about 15 kv.28. The method of embodiment 27, wherein the CE injection voltage is 5kV.29. The method of embodiment 1, wherein a CE injection time is from 5 seconds to 20 seconds.30. The method of embodiment 29, wherein the CE injection time is 10 seconds.31. The method of embodiment 1, wherein a CE separation voltage is 5 kV to 25 kV.32. The method of embodiment 31, wherein the CE separation voltage is 6 kV.33. The method of embodiment 1, wherein a CE separation time is from 10 min to 45 min.34. The method of embodiment 32, wherein the CE separation time is 20 min.35. A method for quantifying a deletion or mutation within an inverted terminal repeat (ITR) sequence of a plasmid, comprising:(a) contacting a sample including the plasmid with a restriction enzyme that cuts within the ITR sequence to generate a digested sample having one or more plasmid fragments; and(b) separating said plasmid fragments using capillary’ electrophoresis (CE) to quantify a deletion or mutation within the ITR sequence.36. The method of embodiment 35, wherein the plasmid lacks a deletion or mutation within the ITR and has two ITR sequences, wherein each ITR sequence contains two recognition sequences and two restriction sites.37. The method of embodiment 36, wherein each ITR sequence contains two recognition sequences specific for cleavage by a restriction enzyme.38. The method of embodiment 35, wherein the restriction enzyme is Xmal, Smal or TspMI.39. The method of embodiment 36, wherein the plasmid without a deletion or mutation within the ITR sequence generates at least four fragments.40. The method of embodiment 39, wherein two of the at least four fragments are about 11 nucleotides long.41. The method of embodiment 39, wherein two of the at least four fragments are about3.4 kb and about 2.6 kb.42. The method of embodiment 35, wherein the plasmid with a deletion or mutation contains one intact ITR sequence including two recognition sequences, each containing two restriction sites.43. The method of embodiment 42, wherein the plasmid with the deletion or mutation can generate two fragments.44. The method of embodiment 35, wherein the ITR sequence is a recombinant adeno- associated virus (AAV) ITR sequence.45. The method of embodiment 35, wherein the ITR sequences comprises between 130 bp and 145 bp.46. The method of embodiment 45, wherein the ITR sequence includes a left ITR sequence and right ITR sequence each comprising 145 nucleotides.47. The method of embodiment 45, wherein either the left ITR sequence or right ITR sequence comprise 141 bp each.48. The method of embodiment 45, wherein either the left ITR sequence or right ITR sequence comprise 130 bp each.49. The method of embodiment 45, wherein the right ITR sequence is truncated.50. The method of embodiment 35, wherein an origin of replication (ORI) is closer to the left ITR.51 . The method of embodiment 35, wherein an ORI is closer to the right ITR.52. The method of embodiment 35, wherein an ORI comprises pUC, R6K or a variant thereof.53. The method of embodiment 35, wherein an ORI is reversed.54. The method of embodiment 35, wherein the plasmid does not contain an antibiotic resistance gene.55. The method of embodiment 35, wherein the plasmid contains an exogenous gene of interest (GOI).56. The method of embodiment 35, wherein a CE capillary length of 30 cm to 60 cm.57. The method of embodiment 56, wherein the CE capillary length is 30 cm.58. The method of embodiment 35, wherein a CE capillary temperature is about 15 °C to about 60 °C.59. The method of embodiment 58, wherein the CE capillary temperature is about 25 °C.60. The method of embodiment 35, wherein a CE injection and separation uses reverse polarih’.61. The method of embodiment 35, wherein a CE injection voltage is 1 kv to about 15 kv.62. The method of embodiment 61, wherein the CE injection voltage is 5kV.63. The method of embodiment 35, wherein the CE injection time is from 5 seconds to 20 seconds.64. The method of embodiment 63, wherein the CE injection time is 10 seconds.65. The method of embodiment 35, wherein the CE separation voltage is 5 kV to 25 kV.66. The method of embodiment 65, wherein the CE separation voltage is 6 kV.67. The method of embodiment 35, wherein the CE separation time is from 10 min to 45 min.68. The method of embodiment 67, wherein the CE separation time is 20 min.69. A method for determining the presence and quantifying a deletion or mutation within an inverted terminal repeat (ITR) sequence of a plasmid, comprising:(a) contacting a sample including the plasmid with a restriction enzyme that cuts within the ITR sequence to generate a digested sample having one or more plasmid fragments; and(b) separating said plasmid fragments using capillary electrophoresis (CE) to determine the presence and quantify a deletion or mutation within the ITR sequence.70. The method of embodiment 69, wherein the plasmid lacks a deletion or mutation within the ITR and has two ITR sequences, wherein each ITR sequence contains two recognition sequences and two restriction sites.71. The method of embodiment 70, wherein each ITR sequence contains two recognition sequences specific for cleavage by a restriction enzyme.72. The method of embodiment 69, wherein the restriction enzyme is Xmal, Smal or TspMI.73. The method of embodiment 70, wherein the plasmid without a deletion or mutation within the ITR sequence generates at least four fragments.74. The method of embodiment 73, wherein two of the at least four fragments are about 11 nucleotides long.75. The method of embodiment 73, wherein two of the at least four fragments are about3.4 kb and about 2.6 kb.76. The method of embodiment 69, wherein the plasmid with a deletion or mutation contains one intact ITR sequence including two recognition sequences, each containing two restriction sites.77. The method of embodiment 76, wherein the plasmid with the deletion of mutation can generate two fragments.78. The method of embodiment 69, wherein the ITR sequence is a recombinant adeno- associated virus (AAV) ITR sequence.79. The method of embodiment 69, wherein the ITR sequences comprises between 130 bp and 145 bp.80. The method of embodiment 79, wherein the ITR sequence includes a left ITR sequence and right ITR sequence each comprising 145 nucleotides.81. The method of embodiment 79, wherein either the left ITR sequence or right ITR sequence comprise 141 bp each.82. The method of embodiment 79, wherein either the left ITR sequence or right ITR sequence comprise 130 bp each.83. The method of embodiment 79, wherein the right ITR sequence is truncated.84. The method of embodiment 69, wherein an origin of replication (ORI) is closer to the left ITR.85. The method of embodiment 69, wherein an ORI is closer to the right ITR.86. The method of embodiment 69, wherein an ORI comprises pUC, R6K or a variant thereof.87. The method of embodiment 69, wherein an ORI is reversed.88. The method of embodiment 69, wherein the plasmid does not contain an antibiotic resistance gene.89. The method of embodiment 69, wherein the plasmid contains an exogenous gene of interest (GOI).90. The method of embodiment 69, wherein a CE capillary length of 30 cm to 60 cm.91. The method of embodiment 90, wherein the CE capillary length is 30 cm.92. The method of embodiment 69, wherein a CE capillary temperature is about 15 °C to about 60 °C.93. The method of embodiment 92, wherein the CE capillary temperature is about 25 °C.94. The method of embodiment 69, wherein a CE injection and separation uses reverse polarity.95. The method of embodiment 69, wherein a CE injection voltage is 1 kv to about 15 kv.96. The method of embodiment 95, wherein the CE injection voltage is 5kV.97. The method of embodiment 69, wherein a CE injection time is from 5 seconds to 20 seconds.98. The method of embodiment 97, wherein the CE injection time is 10 seconds.99. The method of embodiment 69, wherein a CE separation voltage is 5 kV to 25 kV.100. The method of embodiment 99, wherein the CE separation voltage is 6 kV.101. The method of embodiment 69, wherein a CE separation time is from 10 min to 45 min.102. The method of embodiment 101. wherein the CE separation time is 20 min.103. A method for increasing the stability of an inverted terminal repeat (ITR) sequence of a plasmid, comprising: shortening the length of at least one of the ITR sequences by at least four basepairs.104. The method of embodiment 103. wherein the plasmid lacks a deletion or mutation within the ITR and has two ITR sequences, wherein each ITR sequence contains two recognition sequences and two restriction sites.105. The method of embodiment 104, wherein each ITR sequence contains two recognition sequences specific for cleavage by a restriction enzyme.106. The method of embodiment 103. wherein the restriction enzyme is Xmal or Smal.107. The method of embodiment 1 4, wherein the plasmid without a deletion or mutation within the ITR sequence generates at least four fragments w hen contacted by a restriction enzyme.108. The method of embodiment 107. wherein two of the at least four fragments are about 11 nucleotides long.109. The method of embodiment 107, wherein tw o of the at least four fragments are about 3.4 kb and about 2.6 kb.110. The method of embodiment 103. wherein the plasmid with a deletion or mutation contains one intact ITR sequence including two recognition sequences, each containing two restriction sites.111. The method of embodiment 110, wherein the plasmid with the deletion or mutation can generate tw o fragments when contacted by a restriction enzyme.112. The method of embodiment 103. wherein the ITR sequence is a recombinant adeno- associated virus (AAV) ITR sequence.113. The method of embodiment 103, wherein the ITR sequences comprises between 130 bp and 145 bp.114. The method of embodiment 113, wherein either the left ITR sequence or right ITR sequence comprise 141 bp each.115. The method of embodiment 113, wherein either the left ITR sequence or right ITR sequence comprise 130 bp each.116. The method of embodiment 113. wherein the right ITR sequence is truncated.117. The method of embodiment 103, wherein an origin of replication (ORI) is closer to the left ITR.118. The method of embodiment 103. wherein an ORI is closer to the right ITR.119. The method of embodiment 103, wherein an ORI comprises pUC, R6K or a variant thereof.120. The method of embodiment 103. wherein an ORI is reversed.121. The method of embodiment 103, wherein the plasmid does not contain an antibiotic resistance gene.122. The method of embodiment 103. wherein the plasmid contains an exogenous gene of interest (GOI).123. The method of embodiment 103. wherein the plasmid is digested by a restriction enzyme to generate fragments of the plasmid.124. The method of embodiment 123, wherein the fragments of the plasmid are separated using capillary7electrophoresis (CE).125. The method of embodiment 124. wherein a CE capillary' length of 30 cm to 60 cm.126. The method of embodiment 125. wherein the CE capillary’ length is 30 cm.127. The method of embodiment 124, wherein a CE capillary' temperature is about 15 °C to about 60 °C.128. The method of embodiment 125, wherein the CE capillary' temperature is about 25 °C.129. The method of embodiment 124. wherein a CE injection and separation uses reverse polarity'.130. The method of embodiment 124, wherein a CE injection voltage is 1 kv to about 15 kv.131. The method of embodiment 130. wherein the CE injection voltage is 5kV.132. The method of embodiment 124. wherein a CE injection time is from 5 seconds to 20 seconds.133. The method of embodiment 132, wherein the CE injection time is 10 seconds.134. The method of embodiment 124. wherein a CE separation voltage is 5 kV to 25 kV.135. The method of embodiment 134, wherein the CE separation voltage is 6 kV.136. The method of embodiment 124, wherein a CE separation time is from 10 min to 45 min.137. The method of embodiment 136. wherein the CE separation time is 20 min.138. A method for determining the stability of an inverted terminal repeat (ITR) sequence of a plasmid, comprising:(a) shortening the length of at least one of the ITR sequences;(b) contacting a sample including the plasmid with a restriction enzyme that cuts within the ITR sequence to generate a digested sample having one or more plasmid fragments; and(c) separating said plasmid fragments using capillary electrophoresis (CE) to determine the stability7of the ITR sequence.139. The method of embodiment 138. wherein the plasmid lacks a deletion or mutation within the ITR and has two ITR sequences, wherein each ITR sequence contains two recognition sequences and two restriction sites.140. The method of embodiment 139, wherein each ITR sequence contains two recognition sequences specific for cleavage by a restriction enzyme.141. The method of embodiment 138. wherein the restriction enzyme is Xmal. Smal or TspMI.142. The method of embodiment 139, wherein the plasmid without a deletion or mutation within the ITR sequence generates at least four fragments.143. The method of embodiment 142. wherein two of the at least four fragments are about11 nucleotides long.144. The method of embodiment 142, wherein two of the at least four fragments are about 3.4 kb and about 2.6 kb.145. The method of embodiment 138. wherein the plasmid with a deletion or mutation contains one intact ITR sequence including two recognition sequences, each containing two restriction sites.146. The method of embodiment 143, wherein the plasmid with the deletion or mutation can generate two fragments.147. The method of embodiment 138. wherein the ITR sequence is a recombinant adeno- associated virus (AAV) ITR sequence.148. The method of embodiment 138, wherein the ITR sequences comprises between 130 bp and 145 bp.149. The method of embodiment 148, wherein either the left ITR sequence or right ITR sequence comprise 141 bp each.150. The method of embodiment 148, wherein either the left ITR sequence or right ITR sequence comprise 130 bp each.151. The method of embodiment 148. wherein the ITR sequence is truncated.152. The method of embodiment 138, wherein an origin of replication (ORI) is closer to the left ITR.153. The method of embodiment 138. wherein an ORI is closer to the right ITR.154. The method of embodiment 138, wherein an ORI comprises pUC, R6K or a variant thereof.155. The method of embodiment 138. wherein an ORI is reversed.156. The method of embodiment 138, wherein the plasmid does not contain an antibiotic resistance gene.157. The method of embodiment 138. wherein the plasmid contains an exogenous gene of interest (GOI).158. The method of embodiment 138. wherein a CE capillary’ length of 30 cm to 60 cm.159. The method of embodiment 158. wherein the CE capillary length is 30 cm.160. The method of embodiment 138, wherein a CE capillary' temperature is about 15 °C to about 60 °C.161. The method of embodiment 160. wherein the CE capillary' temperature is about 25 °C.162. The method of embodiment 138. wherein a CE injection and separation uses reverse polarity'.163. The method of embodiment 138, wherein a CE injection voltage is 1 kv to about 15 kv.164. The method of embodiment 163. wherein the CE injection voltage is 5kV.165. The method of embodiment 138, wherein a CE injection time is from 5 seconds to 20 seconds.166. The method of embodiment 165, wherein the CE injection time is 10 seconds.167. The method of embodiment 138. wherein a CE separation voltage is 5 kV to 25 kV.168. The method of embodiment 167. wherein the CE separation voltage is 6 kV.169. The method of embodiment 138, wherein a CE separation time is from 10 min to 45 min.170. The method of embodiment 169, wherein the CE separation time is 20 min.

[0270] Embodiment Set 2:1. A method for increasing the stability of an inverted terminal repeat (ITR) sequence of a plasmid, comprising: preparing a plasmid comprising an ITR sequence having a length of about 130 basepairs, wherein the stability is increased as compared to a plasmid comprising an ITR sequence having a length of at least about 145 basepairs.2. The method of embodiment 1, wherein the ITR sequence contains two restriction enzyme sites.3. The method of embodiment 2, wherein the restriction enzyme is Xmal or Smal.4. The method of any one of embodiments 1-3, wherein the plasmid with increased stability lacks a deletion or mutation within the ITR sequence.5. The method of any one of embodiments 2-4. wherein cleavage by the restriction enzyme generates at least four fragments.6. The method of embodiment 5, wherein two of the at least four fragments are about 11 nucleotides long.7. The method of embodiment 5, wherein two of the at least four fragments are about 2.6 kb to about 3.4 kb.8. The method of any one of embodiments 1-4, wherein cleavage by the restriction enzyme generates two fragments.9. The method of any one of embodiments 1-8, wherein the ITR sequence is a recombinant adeno-associated virus (AAV) ITR sequence.10. The method of any one of embodiments 1-9, wherein the ITR sequence is 141 basepairs.11. The method of any one of embodiments 1-9, wherein the ITR sequence is 130 basepairs.12. The method of any one of embodiments 1-11, w herein the plasmid comprises an origin of replication (ORI) that is about 400 to about 600 basepairs away from the ITR sequence.13. The method of any one of embodiments 1-12, wherein the plasmid comprises an ORI that is reverse oriented 3' to 5'.14. The method of embodiment 13, wherein the reverse oriented ORI is about 70 to about 200 basepairs away from the ITR sequence.

[0271] Embodiment Set 3:1. A method for determining the presence of a deletion or mutation within an inverted terminal repeat (ITR) sequence of a plasmid, comprising: contacting a sample including the plasmid with a restriction enzyme that cleaves within the ITR sequence to generate a digested sample having one or more plasmid fragments; and determining the presence of a deletion or mutation within the ITR sequence by separating the plasmid fragments using capillary electrophoresis (CE) and analyzing the number of fragments, wherein a plasmid without a deletion or mutation within the ITR sequence generates more fragments as compared to a plasmid with a deletion or mutation within the ITR sequence.2. The method of embodiment 1, wherein the presence of a deletion or mutation within an inverted terminal repeat (ITR) sequence of a plasmid is quantified by measuring the relative abundance of each fragment peak separated by capillary electrophoresis.3. The method of embodiment 1 or 2, wherein the ITR sequence contains two restriction enzyme sites.4. The method of any one of embodiments 1-3, wherein the restriction enzyme is Xmal, Smal or TspMI.5. The method of any one of embodiments 1 -4, wherein cleavage by the restriction enzyme generates at least four fragments.6. The method of embodiment 5, wherein two of the at least four fragments are about 11 nucleotides long.7. The method of embodiment 5, wherein two of the at least four fragments are about 2.6 kb to about 3.4 kb.8. The method of any one of embodiments 1-4, wherein cleavage by the restriction enzyme generates two fragments.9. The method of any one of embodiments 1-8, wherein the ITR sequence is a recombinant adeno-associated virus (AAV) ITR sequence.10. The method of any one of embodiments 1-9, wherein the ITR sequence is 141 basepairs.11. The method of any one of embodiments 1-9, wherein the ITR sequence is 130 basepairs.12. The method of any one of embodiments 1-9. wherein the ITR sequence is 145 basepairs.13. The method of any one of embodiments 1-12, wherein the plasmid comprises an origin of replication (ORI) that is about 400 to about 600 basepairs away from the ITR sequence.14. The method of any one of embodiments 1-13, wherein the plasmid comprises an ORI that is reverse oriented 3' to 5'.15. The method of embodiment 14, wherein the reverse oriented ORI is about 70 to about 200 basepairs away from the ITR sequence.16. The method of any one of embodiments 1-15, wherein a plasmid with increased stability lacks a deletion or mutation within the ITR sequence.

Claims

CLAIMSWhat is claimed is:

1. A method for determining the presence of a deletion or mutation within an inverted terminal repeat (ITR) sequence of a plasmid, comprising: contacting a sample including the plasmid with a restriction enzy me that cleaves within the ITR sequence to generate a digested sample having one or more plasmid fragments; and determining the presence of a deletion or mutation within the ITR sequence by separating the plasmid fragments using capillary' electrophoresis (CE) and analyzing the number of fragments, wherein a plasmid without a deletion or mutation within the ITR sequence generates more fragments as compared to a plasmid with a deletion or mutation within the ITR sequence.

2. The method of claim 1, wherein the presence of a deletion or mutation within an inverted terminal repeat (ITR) sequence of a plasmid is quantified by measuring the relative abundance of each fragment peak separated by capillary’ electrophoresis.

3. The method of claim 1 or 2, wherein the ITR sequence contains two restriction enzyme sites.

4. The method of any one of claims 1-3, wherein the restriction enzy me is Xmal, Smal or TspMI.

5. The method of any one of claims 1-4, wherein cleavage by the restriction enzyme generates at least four fragments.

6. The method of claim 5, wherein two of the at least four fragments are about 11 nucleotides long.

7. The method of claim 5, wherein two of the at least four fragments are about 2.6 kb to about 3.4 kb.

8. The method of any’ one of claims 1-4, wherein cleavage by the restriction enzyme generates two fragments.

9. The method of any one of claims 1-8, wherein the ITR sequence is a recombinant adeno-associated virus (AAV) ITR sequence.

10. The method of any one of claims 1-9, wherein the ITR sequence is 141 basepairs.

11. The method of any one of claims 1-9, wherein the ITR sequence is 130 basepairs.

12. The method of any one of claims 1-9, wherein the ITR sequence is 145 basepairs.

13. The method of any one of claims 1-12, wherein the plasmid comprises an origin of replication (ORI) that is about 400 to about 600 basepairs away from the ITR sequence.

14. The method of any one of claims 1-13, wherein the plasmid comprises an ORI that is reverse oriented 3' to 5'.

15. The method of claim 14, wherein the reverse oriented ORI is about 70 to about 200 basepairs away from the ITR sequence.

16. The method of any one of claims 1-15, wherein a plasmid with increased stability lacks a deletion or mutation within the ITR sequence.

Images