In the past two decades, adeno-associated virus (AAV) vector manufacturing has made remarkable advancements to meet large-scale production demands for preclinical and clinical trials. In addition, AAV vectors have been extensively studied for their safety and efficacy. In particular, the presence of empty AAV capsids and particles containing “inaccurate” vector genomes in preparations has been a subject of concern. Several methods exist to separate empty capsids from full particles; but thus far, no single technique can produce vectors that are free of empty or partial (non-unit length) capsids. Unfortunately, the exact genome compositions of full, intermediate, and empty capsids remain largely unknown. In this work, we used AAV-genome population sequencing to explore the compositions of DNase-resistant, encapsidated vector genomes produced by two common production pipelines: plasmid transfection in human embryonic kidney cells (pTx/HEK293) and baculovirus expression vectors in Spodoptera frugiperda insect cells (rBV/Sf9). Intriguingly, our results show that vectors originating from the same construct design that were manufactured by the rBV/Sf9 system produced a higher degree of truncated and unresolved species than those generated by pTx/HEK293 production. We also demonstrate that empty particles purified by cesium chloride gradient ultracentrifugation are not truly empty but are instead packaged with genomes composed of a single truncated and/or unresolved inverted terminal repeat (ITR). Our data suggest that the frequency of these “mutated” ITRs correlates with the abundance of inaccurate genomes in all fractions. These surprising findings shed new light on vector efficacy, safety, and how clinical vectors should be quantified and evaluated.
Get full access to this article
View all access options for this article.
References
1.
KuzminDA, ShutovaMV, JohnstonNR, et al.The clinical landscape for AAV gene therapies. Nat Rev Drug Discov, 2021; 20:173–174.
2.
KeelerAM, FlotteTR. Recombinant adeno-associated virus gene therapy in light of luxturna (and zolgensma and glybera): where are we, and how did we get here?. Annu Rev Virol, 2019; 6:601–621.
3.
RodriguesGA, ShalaevE, KaramiTK, et al.Pharmaceutical development of AAV-based gene therapy products for the eye. Pharm Res, 2018; 36:29.
4.
MendellJR, Al-ZaidyS, ShellR, et al.Single-dose gene-replacement therapy for spinal muscular atrophy. N Engl J Med, 2017; 377:1713–1722.
5.
Yla-HerttualaS. Endgame: glybera finally recommended for approval as the first gene therapy drug in the European Union. Mol Ther, 2012; 20:1831–1832.
6.
CotmoreSF, TattersallP. The autonomously replicating parvoviruses of vertebrates. Adv Virus Res, 1987; 33:91–174.
7.
LusbyE, FifeKH, BernsKI. Nucleotide sequence of the inverted terminal repetition in adeno-associated virus DNA. J Virol, 1980; 34:402–409.
8.
BernsKI. The unusual properties of the AAV inverted terminal repeat. Hum Gene Ther, 2020; 31:518–523.
9.
SamulskiRJ, BernsKI, TanM, et al.Cloning of adeno-associated virus into pBR322: rescue of intact virus from the recombinant plasmid in human cells. Proc Natl Acad Sci U S A, 1982; 79:2077–2081.
10.
SamulskiRJ, SrivastavaA, BernsKI, et al.Rescue of adeno-associated virus from recombinant plasmids: gene correction within the terminal repeats of AAV. Cell, 1983; 33:135–143.
11.
ZhouQ, TianW, LiuC, et al.Deletion of the B-B′ and C-C′ regions of inverted terminal repeats reduces rAAV productivity but increases transgene expression. Sci Rep, 2017; 7:5432.
12.
SavyA, DickxY, NauwynckL, et al.Impact of inverted terminal repeat integrity on rAAV8 production using the Baculovirus/Sf9 cells system. Hum Gene Ther Methods, 2017; 28:277–289.
13.
WilmottP, LisowskiL, AlexanderIE, et al.A user's guide to the inverted terminal repeats of adeno-associated virus. Hum Gene Ther Methods, 2019; 30:206–213.
14.
BulchaJT, WangY, MaH, et al.Viral vector platforms within the gene therapy landscape. Signal Transduct Target Ther, 2021; 6:53.
15.
ClementN, GriegerJC. Manufacturing of recombinant adeno-associated viral vectors for clinical trials. Mol Ther Methods Clin Dev, 2016; 3:16002.
16.
GriegerJC, SoltysSM, SamulskiRJ. Production of recombinant adeno-associated virus vectors using suspension HEK293 cells and continuous harvest of vector from the culture media for GMP FIX and FLT1 clinical vector. Mol Ther, 2016; 24:287–297.
17.
UrabeM, DingC, KotinRM. Insect cells as a factory to produce adeno-associated virus type 2 vectors. Hum Gene Ther, 2002; 13:1935–1943.
18.
SmithRH, LevyJR, KotinRM. A simplified baculovirus-AAV expression vector system coupled with one-step affinity purification yields high-titer rAAV stocks from insect cells. Mol Ther, 2009; 17:1888–1896.
19.
MietzschM, GrasseS, ZurawskiC, et al.OneBac: platform for scalable and high-titer production of adeno-associated virus serotype 1–12 vectors for gene therapy. Hum Gene Ther, 2014; 25:212–222.
20.
KurasawaJH, ParkA, SowersCR, et al.Chemically defined, high-density insect cell-based expression system for scalable AAV vector production. Mol Ther Methods Clin Dev, 2020; 19:330–340.
21.
KondratovO, MarsicD, CrossonSM, et al.Direct head-to-head evaluation of recombinant adeno-associated viral vectors manufactured in human versus insect cells. Mol Ther, 2017; 25:2661–2675.
22.
Penaud-BudlooM, FrancoisA, ClementN, et al.Pharmacology of recombinant adeno-associated virus production. Mol Ther Methods Clin Dev, 2018; 8:166–180.
23.
LecomteE, TournaireB, CogneB, et al.Advanced characterization of DNA molecules in rAAV vector preparations by single-stranded virus next-generation sequencing. Mol Ther Nucleic Acids, 2015; 4:e260.
24.
MaynardLH, SmithO, TilmansNP, et al.Fast-Seq: a simple method for rapid and inexpensive validation of packaged single-stranded adeno-associated viral genomes in academic settings. Hum Gene Ther Methods, 2019; 30:195–205.
25.
GuerinK, RegoM, BourgesD, et al.A novel next-generation sequencing and analysis platform to assess the identity of recombinant adeno-associated viral preparations from viral DNA extracts. Hum Gene Ther, 2020; 31:664–678.
26.
TaiPWL, XieJ, FongK, et al.Adeno-associated virus genome population sequencing achieves full vector genome resolution and reveals human-vector chimeras. Mol Ther Methods Clin Dev, 2018; 9:130–141.
27.
RadukicMT, BrandtD, HaakM, et al.Nanopore sequencing of native adeno-associated virus (AAV) single-stranded DNA using a transposase-based rapid protocol. NAR Genom Bioinform, 2020; 2:lqaa074.
28.
TranNT, HeinerC, WeberK, et al.AAV-genome population sequencing of vectors packaging CRISPR components reveals design-influenced heterogeneity. Mol Ther Methods Clin Dev, 2020; 18:639–651.
29.
IbraheimR, TaiPWL, MirA, et al.Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo. Nat Commun, 2021; 12:6267.
30.
SamulskiRJ, ChangLS, ShenkT. A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication. J Virol, 1987; 61:3096–3101.
31.
Penaud-BudlooM, LecomteE, Guy-DucheA, et al.Accurate identification and quantification of DNA species by next-generation sequencing in adeno-associated viral vectors produced in insect cells. Hum Gene Ther Methods, 2017; 28:148–162.
32.
AyusoE, BlouinV, LockM, et al.Manufacturing and characterization of a recombinant adeno-associated virus type 8 reference standard material. Hum Gene Ther, 2014; 25:977–987.
33.
D'CostaS, BlouinV, BroucqueF, et al.Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR. Mol Ther Methods Clin Dev, 2016; 5:16019.
34.
ZhaoH, GhirlandoR, PiszczekG, et al.Recorded scan times can limit the accuracy of sedimentation coefficients in analytical ultracentrifugation. Anal Biochem, 2013; 437:104–108.
35.
SchuckP. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys J, 2000; 78:1606–1619.
36.
BrautigamCA. Calculations and publication-quality illustrations for analytical ultracentrifugation data. Methods Enzymol, 2015; 562:109–133.
37.
JosephS, RussellD. Molecular cloning: a laboratory manual. In: Alkaline Agarose Gel Electrophoresis. New York: Cold Spring Harbor Laboratory Press, 2012:636–666.
38.
LecomteE, LegerA, Penaud-BudlooM, et al.Single-stranded DNA virus sequencing (SSV-Seq) for characterization of residual DNA and AAV vector genomes. Methods Mol Biol, 2019; 1950:85–106.
39.
AfganE, SloggettC, GoonasekeraN, et al.Genomics virtual laboratory: a practical bioinformatics workbench for the cloud. PLoS One, 2015; 10:e0140829.
40.
LiH. Aligning Sequence Reads, Clone Sequences and Assembly Contigs with BWA-MEM. Oxford: Oxford University Press, 2013.
XieJ, MaoQ, TaiPWL, et al.Short DNA hairpins compromise recombinant adeno-associated virus genome homogeneity. Mol Ther, 2017; 25:1363–1374.
46.
LecomteE, SaleunS, BolteauM, et al.The SSV-Seq 2.0 PCR-free method improves the sequencing of adeno-associated viral vector genomes containing GC-rich regions and homopolymers. Biotechnol J, 2021; 16:e2000016.
47.
WangD, TaiPWL, GaoG. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov, 2019; 18:358–378.
48.
GalibertL, SavyA, DickxY, et al.Origins of truncated supplementary capsid proteins in rAAV8 vectors produced with the baculovirus system. PLoS One, 2018; 13:e0207414.
49.
CecchiniS, ViragT, KotinRM. Reproducible high yields of recombinant adeno-associated virus produced using invertebrate cells in 0.02- to 200-liter cultures. Hum Gene Ther, 2011; 22:1021–1030.
50.
LevyHC, BowmanVD, GovindasamyL, et al.Heparin binding induces conformational changes in Adeno-associated virus serotype 2. J Struct Biol, 2009; 165:146–156.
51.
ZhangJ, YuX, GuoP, et al.Satellite subgenomic particles are key regulators of adeno-associated virus life cycle. Viruses, 2021; 13:1185.
52.
WrightJF. Codon modification and PAMPs in clinical AAV vectors: the tortoise or the hare?. Mol Ther, 2020; 28:701–703.
53.
WrightJF. Quantification of CpG motifs in rAAV genomes: avoiding the toll. Mol Ther, 2020; 28:1756–1758.
54.
XiangZ, KurupatiRK, LiY, et al.The effect of CpG sequences on capsid-specific CD8(+) T cell responses to AAV vector gene transfer. Mol Ther, 2020; 28:771–783.
55.
BertoliniTB, ShirleyJL, ZolotukhinI, et al.Effect of CpG depletion of vector genome on CD8(+) T cell responses in AAV gene therapy. Front Immunol, 2021; 12:672449.
PanX, YueY, BoftsiM, et al.Rational engineering of a functional CpG-free ITR for AAV gene therapy. Gene Ther, 2021 Oct 6 [Epub ahead of print]; DOI: 10.1038/s41434-021-00296-0.
58.
ChanYK, WangSK, ChuCJ, et al.Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses. Sci Transl Med, 2021; 13:eabd3438.
59.
JacobA, BrunL, Jimenez GilP, et al.Homologous recombination offers advantages over transposition-based systems to generate recombinant baculovirus for adeno-associated viral vector production. Biotechnol J, 2021; 16:e2000014.
60.
PijlmanGP, van den BornE, MartensDE, et al.Autographa californica baculoviruses with large genomic deletions are rapidly generated in infected insect cells. Virology, 2001; 283:132–138.
61.
GiriL, FeissMG, BonningBC, et al.Production of baculovirus defective interfering particles during serial passage is delayed by removing transposon target sites in fp25k. J Gen Virol, 2012; 93:389–399.
62.
OkanoK, VanarsdallAL, MikhailovVS, et al.Conserved molecular systems of the Baculoviridae. Virology, 2006; 344:77–87.
McCartyDM, FuH, MonahanPE, et al.Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo. Gene Ther, 2003; 10:2112–2118.
66.
WangZ, MaHI, LiJ, et al.Rapid and highly efficient transduction by double-stranded adeno-associated virus vectors in vitro and in vivo. Gene Ther, 2003; 10:2105–2111.
67.
MaurerAC, WeitzmanMD. Adeno-associated virus genome interactions important for vector production and transduction. Hum Gene Ther, 2020; 31:499–511.
68.
YanZ, ZakR, ZhangY, et al.Inverted terminal repeat sequences are important for intermolecular recombination and circularization of adeno-associated virus genomes. J Virol, 2005; 79:364–379.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
