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. 2022 Jun;29(6):333-345.
doi: 10.1038/s41434-021-00296-0. Epub 2021 Oct 6.

Rational engineering of a functional CpG-free ITR for AAV gene therapy

Affiliations

Rational engineering of a functional CpG-free ITR for AAV gene therapy

Xiufang Pan et al. Gene Ther. 2022 Jun.

Abstract

Inverted terminal repeats (ITRs) are the only wild-type components retained in the genome of adeno-associated virus (AAV) vectors. To determine whether ITR modification is a viable approach for AAV vector engineering, we rationally deleted all CpG motifs in the ITR and examined whether CpG elimination compromises AAV-vector production and transduction. Modified ITRs were stable in the plasmid and maintained the CpG-free nature in purified vectors. Replacing the wild-type ITR with the CpG-free ITR did not affect vector genome encapsidation. However, the vector yield was decreased by approximately 3-fold due to reduced vector genome replication. To study the biological potency, we made micro-dystrophin (μDys) AAV vectors carrying either the wild-type ITR or the CpG-free ITR. We delivered the CpG-free μDys vector to one side of the tibialis anterior muscle of dystrophin-null mdx mice and the wild-type μDys vector to the contralateral side. Evaluation at four months after injection showed no difference in the vector genome copy number, microdystrophin expression, and muscle histology and force. Our results suggest that the complete elimination of the CpG motif in the ITR does not affect the biological activity of the AAV vector. CpG-free ITRs could be useful in engineering therapeutic AAV vectors.

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Figures

Figure 1.
Figure 1.. Design of the version 1 CpG-free ITR.
A, Schematic outline of the AAV vector. The expression cassette is composed of a promoter, a transgene, and a poly-adenylation (pA) signal. In an AAV vector, the expression cassette is flanked by two ITRs. The 5’ and 3’ ITRs are highlighted by the dotted boxes. B, Two-dimensional drawing of the 5’-ITR in the flop configuration. The sequence is based on Srivastava et al. Journal of Virology 45(2):555–564, 1983. The AAV ITR is divided into four regions, including the A/A’ stem (sequence A and its complementary sequence A’), B/B’ arm (sequence B, its complementary sequence B’ and three intervening thymine nucleotides between sequences B and B’), C/C’ arm (sequence C, its complementary sequence C’ and three intervening adenine nucleotides between sequences C and C’), and D-sequence (underlined). In addition, there is an unpaired thymidine between the B/B’ and C/C’ arm. Gray letters, nucleotides deleted in the AAV vector. Blue letters, Rep-binding element (RBE). The RBE is a 22-bp sequence (5’-CAGTGAGCGAGCGAGCGCGCAG) as reported in Ryan et al. Journal of Virology 70(3):1542–1553, 1996. The core RBE sequence (blue box) consists of a 10-bp sequence (5’-GCGAGCGAGC). Green letters, the second Rep-binding element (RBE’). The RBE’ is a 5-base sequence (5’-CTTTG) as reported in Brister and Muzyczka Journal of Virology 74(17): 7762–7771, 2000. Red letters, nucleotides in the version 1 CpG-free ITR. Arrowhead, terminal resolution site (trs). Insert, terminology explanation. C, Two-dimensional drawing of the 3’-ITR in the flop configuration. Color coding is the same as in panel B. Purple letters, the core sequence of the trs. The trs core sequence consists of 5’-GT/TGGCC (the forward-slash indicts the nicking site). Insert, terminology explanation. RBE, RBE core sequence, and RBE’ are as in panel B. Nucleotides modified in the CpG-free ITR are marked in red color. D, Alignment of the 3’-ITR of the CpG-free ITR (CpG-free 1) and 3’-ITR of AAV1, 2, 3, 4, 6, and 7. Black dots, nucleotides that are conserved in the 3’-ITR of AAV1, 2, 3, 4, 6, and 7. Asterisks, nucleotides that are conserved in all the listed ITRs. Bold black letters, nucleotides in the AAV2 ITR that are modified in the CpG-free ITR. Red letters, nucleotides modified in the CpG-free ITR. Blue box, the GAGC motif in sequence A’ and the corresponding GCTC motif in sequence A. Green box, the GAGT motif in sequence A’ and the corresponding ACTC motif in sequence A. Dash, nucleotides absent in the AAV vector. Underlined black letters, AAV2 ITR Rep-binding element. Underlined italic blue letters, AAV2 ITR terminal resolution site (trs). Underlined italic purple letters, the second Rep-binding element (RBE’) of the AAV2 ITR. Arrows, nucleotides in B/B’ and C/C’ arms that are not detected in the corresponding position of the ITR from AAV1, 2, 3, 4, 6, and 7.
Figure 2.
Figure 2.. Confirmation of the CpG-free ITR in the cis-plasmid.
A, Nucleotide sequence of the wild-type ITR at the 5’-end of the vector genome (5’-ITR). A, A’, B, B’, C, C’ and D sequences are marked by boxes. The CpG motifs are underlined. B, Nucleotide sequence of the CpG-free ITR at the 5’-end of the vector genome. The mutated nucleotides are highlighted in red. C, Chromatogram view of the sequencing result of the 5’-ITR of the cis-plasmid that carries the CpG-free ITR. Arrows, nucleotides in the CpG-free ITR that are different from those of the wild-type ITR. D, Nucleotide sequence of the wild-type ITR at the 3’-end of the vector genome (3’-ITR). A, A’, B, B’, C, C’ and D sequences are marked by boxes. The CpG motifs are underlined. E, Nucleotide sequence of the CpG-free ITR at the 3’-end of the vector genome. The mutated nucleotides are highlighted in red. F, Chromatogram view of the sequencing result of the 3’-ITR of the cis-plasmid that carries the CpG-free ITR. Arrows, nucleotides in the CpG-free ITR that are different from those of the wild-type ITR.
Figure 3.
Figure 3.. SMRT sequencing evaluation of the ITR sequence in the purified AAV vector.
A, Integrative genomic viewer (IGV) display of SMRT reads from the 5’-ITR of vectors that carry the wild-type ITR. B, IGV display of SMRT reads from the 3’-ITR of vectors that carry the wild-type ITR. C, IGV display of SMRT reads from the 5’-ITR of vectors that carry the CpG-free ITR. D, IGV display of SMRT reads from the 3’-ITR of vectors that carry the CpG-free ITR. Each line represents a single read. Reads mapping on the plus strand are in red, and reads mapping on the minus strand are in blue. The references reflect the flop configurations. Base mismatches are shown as colored bases, inserts are indicated in purple Is, and deletions are shown as dashes. Reads are displayed with soft-clipping on to show the A domain terminus not included in the original reference. Flip-oriented reads are notable by mismatches from the flop reference at positions demarcated by black arrows. Flip reads are indicated in cyan, flop reads in magenta.
Figure 4.
Figure 4.. Quantitative evaluation of AAV production.
A, Quantification of the vector genome yield. *, p<0.05. B, Representative AAV capsid dot blot. The blot was probed with an antibody that only recognizes intact AAV9 particles. C, Quantification of the vector capsid yield. **, p<0.01. D, Representative transmission electron microscopy images of the CpG-free ITR vector. Arrow, a fully packaged AAV particle. Arrowhead, an empty AAV particle. E, Representative transmission electron microscopy images of the wild-type ITR vector. Arrow, a fully packaged AAV particle. Arrowhead, an empty AAV particle. F, Quantification of empty particles. Each data point represents the quantification result from one field at the 25,000x magnification. For the wild-type ITR vector, a total of 48 fields were quantified. For the CpG-free ITR vector, a total of 25 fields were quantified.
Figure 5.
Figure 5.. Evaluation of vector genome replication during AAV production.
Human 293FT cells were co-transfected with the cis-plasmid (pXP15 or pXP24), pHelper, and pRepCap. Hirt DNA was harvested at the indicated time points and analyzed by Southern blot using homologous genomic probes. A, Representative Southern blot using Hirt DNA harvested at 2 and 48 hours post-transfection (lanes 2 to 7). Marker (lane 1), a 5-kb band from SacI and SalI double digestion of pXP15 or pXP24 to indicate the full-length vector genome. B, A biological replicate Southern blot using Hirt DNA harvested at 2, 24, and 48 hours post-transfection (lanes 2 to 9). Lane 1 is undigested plasmid pXP24. Arrow, monomer replication form (mRF), and dimer replication form (dRF). White arrowhead, the cis-plasmid bands that are larger than the size of mRF. DpnI digestion completely removed these bands. Black arrowhead, the cis-plasmid band that co-migrated with mRF.
Figure 6.
Figure 6.. The CpG-free ITR optimization and the yield from the vector carrying the modified CpG-free ITR.
A, Alignment of the 3’-ITR from B/B’ and C/C’ arm optimized CpG-free ITR (CpG-free 2) and 3’-ITR from AAV1, 2, 3, 4, 6, and 7. Black dots, nucleotides that are conserved in the 3’-ITR of AAV1, 2, 3, 4, 6, and 7. Asterisks, nucleotides that are conserved in all the ITRs. Bold black letters, nucleotides in the AAV2 ITR that are modified in the CpG-free ITR. Red letters, nucleotides that are different from AAV2 ITR. Yellow highlighted letters, nucleotides that are different from the version 1 CpG-free ITR. Yellow letters, thymine in AAV3 and AAV4 that are shared by the version 2 CpG-free ITR. Underlined black letters, AAV2 ITR Rep-binding element. Underlined italic blue letters, AAV2 ITR terminal resolution site (trs). Underlined italic purple letters, the second Rep-binding element (RBE’) of the AAV2 ITR. Blue box, the conserved GAGY motif in sequence A’ and the corresponding RCTC motif in sequence A. Dash, nucleotides absent in the AAV vector. B, Quantification of the vector genome yield from the culture medium. *, p<0.05. C, quantification of the vector genome yield from cell lysate. **, p<0.01.
Figure 7.
Figure 7.. Evaluation of micro-dystrophin expression.
A, Representative dystrophin immunofluorescence staining and HE staining micrographs from the tibialis anterior (TA) muscle of dystrophin-null mdx mice that were injected with the CpG-free micro-dystrophin (μDys) vector (left panels) and the wild-type μDys vector (middle panels). The TA muscle from an age and sex-matched un-injected mdx mouse was included as the control (right panels). Scale bar applies to all images. B, Quantification of dystrophin positive myofibers. C, Western blot evaluation of 143 kD μDys from TA muscles of CpG-free μDys vector and wild-type μDys vector treated mice. Alpha-tubulin (50 kD) is used as the loading control. D, Quantification of western blot μDys expression. E, Quantification of the vector genome copy number in the TA muscle.
Figure 8.
Figure 8.. Evaluation of centronucleation and myofiber size distribution.
A, The percentage of myofibers that contained centrally localized nuclei in the mdx muscle treated with the CpG-free μDys vector and the wild-type μDys vector. B, The distribution of the percentage of myofibers at different minimum Feret diameters in 616 myofibers from the CpG-free μDys vector treated muscle (n= 6 muscles, 80 to 131 myofibers per muscle) and 712 myofibers from the wild-type μDys vector treated muscle (n= 6 muscles, 87 to 135 myofibers per muscle).
Figure 9.
Figure 9.. Quantitative evaluation of the tibialis anterior muscle contractility from mdx mice that were treated with CpG-free and the wild-type μDys vectors.
A, TA muscle weight. B, TA muscle cross-sectional area (CSA). C, Absolute twitch force (Pt). D, Specific twitch force (sPt). E, Absolute tetanic force (Po). F, Specific tetanic force (sPo). G, Force-frequency relationship. H, Eccentric contraction profiles.

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