Short DNA Hairpins Compromise Recombinant Adeno-Associated Virus Genome Homogeneity

doi:10.1016/j.ymthe.2017.03.028

. 2017 Jun 7;25(6):1363-1374.

doi: 10.1016/j.ymthe.2017.03.028. Epub 2017 Apr 24.

Short DNA Hairpins Compromise Recombinant Adeno-Associated Virus Genome Homogeneity

Jun Xie¹, Qin Mao², Phillip W L Tai³, Ran He⁴, Jianzhong Ai⁵, Qin Su⁴, Ye Zhu⁶, Hong Ma⁴, Jia Li⁷, Shoufang Gong⁷, Dan Wang³, Zhen Gao⁷, Mengxin Li⁴, Li Zhong⁷, Heather Zhou⁸, Guangping Gao⁹

Affiliations

¹ Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Viral Vector Core, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA 01605, USA.
² Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Thoracic Cancer, Cancer Center, West China Hospital, West China School of Clinical Medicine, Sichuan University, Chengdu, Sichuan 610000, China.
³ Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA 01605, USA.
⁴ Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Viral Vector Core, University of Massachusetts Medical School, Worcester, MA 01605, USA.
⁵ Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Urology Department, West China Hospital, Sichuan University, Chengdu, Sichuan 610000, China.
⁶ Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Cardiovascular Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610000, China.
⁷ Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA.
⁸ Genetics and Pharmacogenomics, Merck Research Laboratory, Kenilworth, NJ 07033, USA. Electronic address: hui_zhou@merck.com.
⁹ Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Viral Vector Core, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA 01605, USA; State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610000, China. Electronic address: guangping.gao@umassmed.edu.

PMID: 28462820
PMCID: PMC5474962
DOI: 10.1016/j.ymthe.2017.03.028

Short DNA Hairpins Compromise Recombinant Adeno-Associated Virus Genome Homogeneity

Jun Xie et al. Mol Ther. 2017.

. 2017 Jun 7;25(6):1363-1374.

doi: 10.1016/j.ymthe.2017.03.028. Epub 2017 Apr 24.

Authors

Affiliations

¹ Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Viral Vector Core, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA 01605, USA.
² Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Thoracic Cancer, Cancer Center, West China Hospital, West China School of Clinical Medicine, Sichuan University, Chengdu, Sichuan 610000, China.
³ Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA 01605, USA.
⁴ Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Viral Vector Core, University of Massachusetts Medical School, Worcester, MA 01605, USA.
⁵ Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Urology Department, West China Hospital, Sichuan University, Chengdu, Sichuan 610000, China.
⁶ Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Cardiovascular Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610000, China.
⁷ Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA.
⁸ Genetics and Pharmacogenomics, Merck Research Laboratory, Kenilworth, NJ 07033, USA. Electronic address: hui_zhou@merck.com.
⁹ Horae Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA; Viral Vector Core, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA 01605, USA; State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610000, China. Electronic address: guangping.gao@umassmed.edu.

PMID: 28462820
PMCID: PMC5474962
DOI: 10.1016/j.ymthe.2017.03.028

Abstract

Short hairpin (sh)RNAs delivered by recombinant adeno-associated viruses (rAAVs) are valuable tools to study gene function in vivo and a promising gene therapy platform. Our data show that incorporation of shRNA transgenes into rAAV constructs reduces vector yield and produces a population of truncated and defective genomes. We demonstrate that sequences with hairpins or hairpin-like structures drive the generation of truncated AAV genomes through a polymerase redirection mechanism during viral genome replication. Our findings reveal the importance of genomic secondary structure when optimizing viral vector designs. We also discovered that shDNAs could be adapted to act as surrogate mutant inverted terminal repeats (mTRs), sequences that were previously thought to be required for functional self-complementary AAV vectors. The use of shDNAs as artificial mTRs opens the door to engineering a new generation of AAV vectors with improved potency, genetic stability, and safety for both preclinical studies and human gene therapy.

Keywords: adeno-associated virus; genome homogeneity; replication template switching; self-complementary AAV; short hairpin DNA; short hairpin RNA; viral genome truncation.

Published by Elsevier Inc.

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Figures

**Figure 1**
Positioning of shRNA Cassettes within Self-Complementary AAV Constructs Impacts Vector Yield (A) Vector yield (genome copies [GCs]) comparison of independent self-complementary AAV8 preparations with (n = 15) or without (n = 11) shRNA cassettes designed proximal to the wtTR. (B) Schematic of self-complementary AAV plasmids consisting of a CMV enhancer/chicken β-actin promoter (CB), an EGFP reporter gene, and a beta-globin polyA sequence (PA). The shRNA cassettes targeting Apob, driven by the H1 promoter, or targeting the Firefly luciferase gene (Fluc), driven by the U6 promoter, were inserted adjacent to the mTR (m-R and m-F), within the intron (Intron-R and Intron-F), or adjacent to the wtTR (Wt-R and Wt-F). (C) Vectors depicted in (B) were packaged into AAV9 capsids and assessed for yield by qPCR using an EGFP primer/probe set. Constructs carrying the same shRNA cassette were packaged and titrated as a set (at the same time) to ensure fair comparisons. The two sets of constructs (U6-shFluc and H1-shApob) were packaged at different times. (D) Vector yield comparison of independent self-complementary AAV9 preparations that lack an shRNA cassette (n = 5) or with different shRNAs (n = 6) positioned proximal to the mTR (m-R). N.S., not significant.

**Figure 2**
EGFP Transgene Expression and Profiling of rAAV Genomes in the Liver of Mice Receiving Self-Complementary AAV-shApob Vectors The self-complementary AAV9 vectors carrying EGFP transgene and shApob in different positions (5 × 10¹³ GCs/kg) were injected by tail vein into 6- to 8-week-old C57BL/6 mice. Mice were sacrificed 3 weeks later. (A) EGFP expression in livers as determined by fluorescence microscopy. Scale bar, 100 μM. Quantitation of vector genomes by qPCR using EGFP primer/probe is displayed above each image to demonstrate the detection of comparable vector genomes after transduction using equal dosages. Values are mean ± SD (n = 6). (B) Southern blot analysis of rAAV molecular forms isolated from livers using an EGFP sequence probe. Liver DNAs were digested with EcoRI (non-cutter) or MscI (single cutter within the wtTR) prior to hybridization. The banding patterns of the heterogeneous vector populations are hypothesized to relate to the vector genome sizes spanning from the wtTR or the MscI cut site to the mTR or the shRNA cassette for each construct. Circularized AAV genomes migrate faster than their linear counterparts. Each test vector is diagrammed below, displaying the molecular length (kb) between the shRNA cassette (sh, orange loop) and the wtTR/MscI cut site. Black arrows, linear intact rAAV genomes; purple arrows, circular intact rAAVs; asterisks, linear truncated genomes; black arrowheads, circular truncated populations; magenta arrows, MscI linearized intact rAAVs; white arrowheads, MscI linearized truncated rAAVs. See also Figures S1 and S2.

**Figure 3**
Genome Populations in rAAV Vectors Containing shRNA Cassettes (A) Agarose gel analysis of self-complementary AAV vector genomes carrying shApob, driven by the H1 promoter, or shFluc, driven by the U6 promoter. Cassettes were each tested in the six positions/orientations as illustrated in Figure 1B. (B) AAV vector genomes (AAV8, AAV9, AAVrh10, and AAV2) carrying intronic shRNA cassettes designed to target different genes. (C) AAV9 genomes carrying different shRNA cassettes inserted between the polyA and the wtTR. (D) AAV6 and AAV8 genomes harboring shRNA cassettes inserted between the mTR and the CB promoter. Vector DNA equivalents of 0.1–1 × 10¹¹ GC viral genomes were assessed by agarose gel electrophoresis. sh-1 to sh-26 represents 26 different shDNA sequences. The distances between the shDNA region and the wtTR are indicated above each vector diagram. In (C), vector sh-24 contains the H1 promoter and vectors sh-25 and sh-26 contain the U6 promoter. Due to the size difference between the H1 and U6 promoters, the distances between shRNA and wtTR are 0.4 and 0.5 kb, respectively. Full-length (black arrows) and shRNA-related truncated (purple arrows) genome bands are indicated among other additional smaller bands. (E) The molar ratio of truncated genomes to full-length genomes in AAV vectors carrying shDNA at different positions/orientations. Ratios were calculated by normalizing their band intensities by densitometry to their molecular sizes. The ratios of truncated to full-length genomes of Wt-F (n = 5), Wt-R (n = 5), Intron-F (n = 12), Intron-R (n = 2), m-F (n = 9), and m-R (n = 2) preparations are reported on a log scale. Values are mean ± SD. See also Figure S3.

**Figure 4**
Characterization of Truncated rAAV Genomes (A) Model of rAAV genome replication detoured by a short DNA hairpin. (B) DNAs extracted from rAAV vectors were examined on an alkaline agarose gel. Red arrows indicate bands related to full-length genomes, while blue arrows denote the truncated genomes caused by the shDNA sequences in each preparation. (C) Diagram showing the strategy for SMRT sequencing library preparation and data processing. The specifics of this strategy are detailed in the Materials and Methods. (D) Model-guided sequence prediction of truncated AAV genomes. Functional segments of the mTR are displayed as follows: Rep-binding element (RBE), the B-B’ hairpin, and the C-C’ hairpin. “A” represents the replicated A domain in the vector genome. The model predicts that the stem of shDNA in the Intron-R vector may interfere with replication, resulting in a switch in replication templates to the newly synthesized daughter strand (middle panels). As a result, sequences spanning the shRNA sense strand, the H1 promoter, the EGFP transgene, and finally the wtTR sequence are re-replicated to form truncated genomes. Intron-F vector genomes are re-directed in the same fashion to generate genomes that lack the H1 promoter sequence and beyond (right panel). (E) SMRT sequencing reads aligned to custom references that represent self-complementary sequence resulting from template-switching events at the mTR (top panel) and the shApob-encoding sequences (middle panel, Intron-F; and bottom panel, Intron-R). See also Figures S4 and S5.

**Figure 5**
Characterization of shAAV Genomes and In Vivo Evaluation of shAAV Vectors (A) Schematic of pCis constructs used for AAV production. The mTR was removed from vector constructs to assess the ability of shDNA sequences to create double-stranded shAAV vectors. (B) The predicted sizes of packaged genomes were calculated from the base pair lengths between shDNA sequences and wtTR. (C) Viral genome DNA from purified vectors (∼1.0 × 10¹⁰ GCs) in native (left panel) and alkaline (right panel) agarose gels. (D) EGFP expression in livers of adult mice 3 weeks after intravenous injection of rAAV (1.6 × 10¹³ GCs/kg). (E) Southern blot analysis of EcoRI- or MscI-digested liver DNA using an EGFP probe. The MscI site is denoted in (A). Small black arrows, linear rAAV genomes; purple arrows, circular rAAVs; magenta arrows, linearized circular rAAVs; white arrowheads, digested linear rAAVs.

**Figure 6**
Characterization of Variable Vector Genomes Generated from shDNA-like Sequences (A) Aggregation plots of alignment termination positions along the *pH1-shApob1.3* construct (top panel) or the *scAAV-EGFP* construct (bottom panel), as assessed by direct SMRT sequencing of rAAV genomes. Positional tags were distributed into intervals of 10-nt bins and the density of tags was plotted along the *H1-shApob1.3* vector sequence. Peaks indicate regions along the genome where termination hotspots occur. Due to an inherent size bias of SMRT sequencing for smaller molecules, sequences identified by SMRT analysis only reveal the diversity of truncated genome types and do not directly reflect their relative abundances. Sequences of discovered hotspots are flanked by inverted repeats (IRs). Two are present in the CMV enhancer (inverted repeat-1 and inverted repeat-2), one in the CB promoter (inverted repeat-3), and one in the EGFP reporter gene (inverted repeat-4). (B) The predicted secondary structures of IR1–4 using RNA Fold. Bases highlighted in gray indicate inverted repeat sequences. (C) Sequence alignments of rAAV genomes to a reference consisting of self-complementary strands flanking the inverted repeat-3 sequence (top). The bottom panel details the loop sequence that connects the partial CB promoter and its reverse-complement sequence. (D) Southern blot analysis of low-molecular-weight DNAs using an EGFP probe from HEK293 cells after triple transfection with an self-complementary AAV-EGFP vector, a vector that has in place of the wtTR an shDNA sequence (*shDNA*⁺*wtTR*⁻), or a vector that lacks the mTR (*mTR*⁻) (left blot). Diagrams of these vectors are displayed to the right. Agarose gel of vector DNAs from purified rAAV (center) shows the heterogeneity of packaged genomes. (E) EGFP expression of mouse livers 3 weeks post-injection of 1.6 × 10¹³ GCs/kg self-complementary AAV9 or mTR⁻ vectors. Scale bar, 100 μm; n = 4. See also Figures S6 and S7.

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References

1. Handa H., Carter B.J. Adeno-associated virus DNA replication complexes in herpes simplex virus or adenovirus-infected cells. J. Biol. Chem. 1979;254:6603–6610. - PubMed
1. de la Maza L.M., Carter B.J. Molecular structure of adeno-associated virus variant DNA. J. Biol. Chem. 1980;255:3194–3203. - PubMed
1. McLaughlin S.K., Collis P., Hermonat P.L., Muzyczka N. Adeno-associated virus general transduction vectors: analysis of proviral structures. J. Virol. 1988;62:1963–1973. - PMC - PubMed
1. Samulski R.J., Chang L.S., Shenk T. Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J. Virol. 1989;63:3822–3828. - PMC - PubMed
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[1] Handa H., Carter B.J. Adeno-associated virus DNA replication complexes in herpes simplex virus or adenovirus-infected cells. J. Biol. Chem. 1979;254:6603–6610. - PubMed

[2] Handa H., Carter B.J. Adeno-associated virus DNA replication complexes in herpes simplex virus or adenovirus-infected cells. J. Biol. Chem. 1979;254:6603–6610. - PubMed

[3] de la Maza L.M., Carter B.J. Molecular structure of adeno-associated virus variant DNA. J. Biol. Chem. 1980;255:3194–3203. - PubMed

[4] de la Maza L.M., Carter B.J. Molecular structure of adeno-associated virus variant DNA. J. Biol. Chem. 1980;255:3194–3203. - PubMed

[5] McLaughlin S.K., Collis P., Hermonat P.L., Muzyczka N. Adeno-associated virus general transduction vectors: analysis of proviral structures. J. Virol. 1988;62:1963–1973. - PMC - PubMed

[6] McLaughlin S.K., Collis P., Hermonat P.L., Muzyczka N. Adeno-associated virus general transduction vectors: analysis of proviral structures. J. Virol. 1988;62:1963–1973. - PMC - PubMed

[7] Samulski R.J., Chang L.S., Shenk T. Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J. Virol. 1989;63:3822–3828. - PMC - PubMed

[8] Samulski R.J., Chang L.S., Shenk T. Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J. Virol. 1989;63:3822–3828. - PMC - PubMed

[9] Asokan A., Schaffer D.V., Samulski R.J. The AAV vector toolkit: poised at the clinical crossroads. Mol. Ther. 2012;20:699–708. - PMC - PubMed

[10] Asokan A., Schaffer D.V., Samulski R.J. The AAV vector toolkit: poised at the clinical crossroads. Mol. Ther. 2012;20:699–708. - PMC - PubMed

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Short DNA Hairpins Compromise Recombinant Adeno-Associated Virus Genome Homogeneity

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Short DNA Hairpins Compromise Recombinant Adeno-Associated Virus Genome Homogeneity

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