DNA Minicircle Technology Improves Purity of Adeno-associated Viral Vector Preparations

Abstract

Adeno-associated viral (AAV) vectors are considered as one of the most promising delivery systems in human gene therapy. In addition, AAV vectors are frequently applied tools in preclinical and basic research. Despite this success, manufacturing pure AAV vector preparations remains a difficult task. While empty capsids can be removed from vector preparations owing to their lower density, state-of-the-art purification strategies as of yet failed to remove antibiotic resistance genes or other plasmid backbone sequences. Here, we report the development of minicircle (MC) constructs to replace AAV vector and helper plasmids for production of both, single-stranded (ss) and self-complementary (sc) AAV vectors. As bacterial backbone sequences are removed during MC production, encapsidation of prokaryotic plasmid backbone sequences is avoided. This is of particular importance for scAAV vector preparations, which contained an unproportionally high amount of plasmid backbone sequences (up to 26.1% versus up to 2.9% (ssAAV)). Replacing standard packaging plasmids by MC constructs not only allowed to reduce these contaminations below quantification limit, but in addition improved transduction efficiencies of scAAV preparations up to 30-fold. Thus, MC technology offers an easy to implement modification of standard AAV packaging protocols that significantly improves the quality of AAV vector preparations.

Keywords: AAV vectors; DNA impurities; Minicircle; Vector production; Vector quality.

Figure 1

Antibiotic resistance gene (ampR) is tightly associated with adeno-associated viral (AAV) vector particles. (a) Quantification of DNA sequences contained in AAV vector preparations. Lysates of vector-producing cells were purified by discontinuous iodixanol density gradient centrifugation (IDGC). The 40%-phases of three different vector preparations—containing AAV vector particles—were further purified by either affinity chromatography using AVB column (AC-AVB) or Heparin column (AC-Heparin), followed by centrifugal filtration (CF) or gel filtration (GF). At each step of purification, total DNA from aliquots was isolated and analyzed by qPCR using indicated primers. (b) Benzonase protection assay. Two aliquots of AAV vector preparation #3 encoding for eGFP were spiked with 400 ng of a plasmid encoding for the kanamycin/neomycin resistance gene (kanR). One aliquot was treated with Benzonase as described in Materials&Methods section. Total DNA of the treated and an untreated second aliquot was isolated and analyzed by qPCR using indicated primers (Supplementary Table S1). qPCR analyses described in (a) and (b) were performed three times independently.

Figure 2

Replacing vector plasmid by minicircle construct significantly decreases the amount of encapsidated ampR DNA particles. DNA isolated from vector preparations was quantified for ampR sequences by qPCR using specific primers (Supplementary Table S1). # < Limit of quantification. All analyses were performed in parallel for all vector preparations. All analyses were performed three times independently. Differences in ampR content between preparations were assessed using ANOVA (P < 0.0001) and subsequent Tukey post hoc tests.

Figure 3

Replacement of both plasmids by minicircle results in ampR-free vector preparations. DNA isolated from vector preparations was quantified for ampR sequences by qPCR using specific primers (Supplementary Table S1). # < Limit of quantification. Analysis was performed in parallel for all vector preparations. Differences in ampR content between preparations were assessed using ANOVA (P < 0.0001) and subsequent Tukey post hoc tests.

Figure 4

Model of proposed rescue mechanism. Circular plasmid genome is represented as linearized. (a) Schematic representation of AAV2 ITR. The triangle represents the nicking activity of Rep at the trs. (b) Rep nicks at trs and creates a single-strand break. (c) The template strand folds into a hairpin (HP) conformation thus enabling replication along the displaced strand. (d) The newly generated inverted terminal repeat (ITR) folds into the HP conformation which allows for replication into the vector genome sequence. (e) Upon the nicking event at the trs of the second ITR, the single-stranded AAV vector genome is created. (f) If the second ITR is not nicked before the arrival of the replication machinery a large replicon encompassing TEC and backbone sequences is generated. (g) The TEC genome can be rescued by Rep nickase activity which then creates the TEC with one intact ITR and an additional D sequence and the prokaryotic backbone sequences with two ITRs lacking the D sequence. (h) The defect ITR of the TEC genome can be repaired by a panhandle mechanism, thus generating an intact AAV vector genome. RBE: Rep protein binding element, trs: terminal resolution site. Figure adapted from Ward et al.