“D” matters in recombinant AAV DNA packaging
Authors: Junping Zhang, Ping Guo, Yinxia Xu, Patrick L. Mulcrone, R. Jude Samulski, Weidong Xiao
Journal: Molecular Therapy (2021)

To the editor: Unmethylated CpG dinucleotide-based motifs (CpGs) have been documented as pathogen-associated molecular patterns (PAMPs) that bind to Toll-like receptor (TLR)9 pattern recognition receptors and lead to adaptive effector functions that result in a loss of transduced cells.1Faust S.M. Bell P. Cutler B.J. Ashley S.N. Zhu Y. Rabinowitz J.E. Wilson J.M. CpG-depleted adeno-associated virus vectors evade immune detection.J. Clin. Invest. 2013; 123: 2994-3001Crossref PubMed Scopus (130) Google Scholar Vector genome hypomethylation has been recognized as a deficiency of current methods of generation of adeno-associated viral (AAV) vectors.2Wright J.F. Codon Modification and PAMPs in Clinical AAV Vectors: The Tortoise or the Hare?.Mol. Ther. 2020; 28: 701-703Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Although wild-type parvovirus DNA has been shown to be generally hypomethylated3Tóth R. Mészáros I. Hüser D. Forró B. Marton S. Olasz F. Bányai K. Heilbronn R. Zádori Z. Methylation Status of the Adeno-Associated Virus Type 2 (AAV2).Viruses. 2019; 11: 11Crossref Scopus (8) Google Scholar in its replication cycle, one additional source of hypomethylation in recombinant AAV (rAAV) genomes stems from the encapsidation of unreplicated vector genomes, which are often directly derived from bacterial plasmid DNA origin.2Wright J.F. Codon Modification and PAMPs in Clinical AAV Vectors: The Tortoise or the Hare?.Mol. Ther. 2020; 28: 701-703Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Since the mainstay method for clinical AAV production has been based on triple plasmid transfection, understanding the mechanism of the direct packaging of plasmid-derived DNA is necessary to find a solution to this problem. Traditionally, we think of AAV vector production as involving DNA replication. AAV vector genomes are constructed by flanking the expression cassette with two copies of AAV inverted terminal repeat (ITR) as the essential cis elements to facilitate the replication of vector DNA and packaging.4Xiao X. Xiao W. Li J. Samulski R.J. A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle.J. Virol. 1997; 71: 941-948Crossref PubMed Google Scholar A key element in the AAV ITR identified to control such a function is the D-element (“D”), which is a 20-nucleotide sequence that is highly conserved among various AAV serotypes. “D” is a unique region of AAV ITR that is not part of the palindromic sequences. In addition, the “D” is located near the terminal resolution site, which is the initial site for rescue, replication, and packaging.5Snyder R.O. Samulski R.J. Muzyczka N. In vitro resolution of covalently joined AAV chromosome ends.Cell. 1990; 60: 105-113Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 6Wang X.S. Ponnazhagan S. Srivastava A. Rescue and replication signals of the adeno-associated virus 2 genome.J. Mol. Biol. 1995; 250: 573-580Crossref PubMed Scopus (53) Google Scholar, 7Wang X.S. Ponnazhagan S. Srivastava A. Rescue and replication of adeno-associated virus type 2 as well as vector DNA sequences from recombinant plasmids containing deletions in the viral inverted terminal repeats: selective encapsidation of viral genomes in progeny virions.J. Virol. 1996; 70: 1668-1677Crossref PubMed Google Scholar AAV rep genes and capsid genes along with a set of helper virus genes are provided in trans to complement the functionality of the AAV ITR and D-element during vector production. Nevertheless, it is challenging to study the role of D-element function because of the strong secondary structure in AAV ITRs and the necessity of replacing the two copies of D-elements in the AAV genomes simultaneously. We overcame this technical hurdle by utilizing a self-complimentary vector in our study, which allowed a random library of D-sequences to be analyzed in the rAAV packaging process. The analysis of the D-element requirement for rAAV vector encapsidation revealed an astonishing outcome: wild-type AAV (WTAAV) ITR D-sequences were recovered only ∼25% of the time (Figure S1). This suggests that any sequence replacing the D-element (so called “pseudo-D” sequence) could be utilized by rAAV for packaging, indicating a lax requirement for D-sequences in rAAV encapsidation and providing an explanation for the existence of plasmid backbone in the rAAV preparations. Textbooks state that the D-element is essential for WTAAV replication and packaging. However, the results from rAAV packaging and the above studies clearly show a difference between rAAV and WTAAV. We think that the DNA replication differences between the rAAV production system and WTAAV propagation are the primary cause for such a discrepancy. WTAAV propagation usually arises from initial low MOI infection, and successful productive lytic infection from a single virus infection or latent viral genome rescue would occur naturally. This means that viral DNA will have to be amplified extensively before encapsidation. As illustrated in Figure 1A, if a mutant “D” emerged because of replication error or for some other reason, even assuming that this mutant “D” had a modestly lower replication efficiency (say ∼80%), DNA with a mutant “D” would only account for 0.1% of sequences and effectively be eliminated after 30 rounds of replication to reach a low titer of 1E10 genomes from a single genome. Therefore, such mutant D-elements, even with a slight disadvantage in replication, are less likely to survive during WTAAV replication and propagation, thus promoting predominance of the highly conservative D-sequences observed among many different AAV serotypes. However, the packaging process of rAAV vectors is different. As illustrated in Figure 1B, AAV ITRs are mostly symmetric in the vector plasmid used for production. D-elements function to specifically promote the vector genome part of the plasmid to be replicated and packaged. Upon rescue of the vector genome from the plasmid molecule, owing to the symmetry of the ITRs, two DNA fragments are expected to be generated. One is the vector genome containing the D-sequences that would lead to the replication and packaging of the desired rAAV vectors. The other part is the plasmid backbone, which retains all AAV ITR palindromic sequences except the unique D-sequences. Therefore, we asked whether the 20 nucleotides immediately adjacent to the ITRs in the plasmid backbone side could act as alternative “D” sequences, or so called “pseudo-D” elements. Although such pseudo-D sequences have been documented to be deficient in replication, previous observations and our new data (see Figure S2) demonstrate that pseudo-“D”s are still compatible with AAV encapsidation. Indeed, in a typical AAV production system, viral particles with plasmid backbone may represent as much as one-tenth of all viral particles produced.2Wright J.F. Codon Modification and PAMPs in Clinical AAV Vectors: The Tortoise or the Hare?.Mol. Ther. 2020; 28: 701-703Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar,8Wright J.F. Product-Related Impurities in Clinical-Grade Recombinant AAV Vectors: Characterization and Risk Assessment.Biomedicines. 2014; 2: 80-97Crossref PubMed Scopus (66) Google Scholar The high level of contamination with plasmid backbone provides more evidence for limited replication of AAV vector DNA in these production systems. Since WT D is not the dominating or decisive factor to exclude packaging pseudo-D DNA into AAV capsid, the ratio of WT-D DNA and pseudo-D plasmid backbone DNA is therefore dictating the composition of DNA in the packaged capsid, as illustrated in Figure 1B. After rescue from the plasmid DNA, the ratio of pseudo-D DNA and vector DNA is 1:1, without any DNA replication. In this scenario, if pseudo-D DNA were packaged as efficiently as WT-D DNA, the amount of vector with plasmid backbone would be the same as vector DNA with D-sequences. However, if the vector DNA with D-element were to be replicated twice, then the ratio of plasmid backbone to vector DNA in the vector would be 1:3. Such a balance would vary according to the property of the “pseudo-D” that happens to be in the vector DNA plasmid. Since the plasmid backbone is commonly observed at 10% of all genomes in AAV vectors, limited vector DNA replication must have taken place prior to rAAV packaging (if pseudo-D is not as efficient as WT D in packaging, vector DNA actually replicates even less). If, on the other hand, the vector DNA were to be replicated 30 times, vector plasmid DNA would be diluted out and therefore not affect the vector composition. Thus, the lax requirement of D-element in packaging is a major factor contributing to vector composition, as reflected by the considerable level of contamination of plasmid backbone,8Wright J.F. Product-Related Impurities in Clinical-Grade Recombinant AAV Vectors: Characterization and Risk Assessment.Biomedicines. 2014; 2: 80-97Crossref PubMed Scopus (66) Google Scholar which is typically found in vector preparations using plasmids as template. Despite extensive DNase clearing, viral particles containing the bacterial backbone are still observed in even the most highly purified preparations.9Chadeuf G. Ciron C. Moullier P. Salvetti A. Evidence for encapsidation of prokaryotic sequences during recombinant adeno-associated virus production and their in vivo persistence after vector delivery.Mol. Ther. 2005; 12: 744-753Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar,10Salvetti A. Orève S. Chadeuf G. Favre D. Cherel Y. Champion-Arnaud P. David-Ameline J. Moullier P. Factors influencing recombinant adeno-associated virus production.Hum. Gene Ther. 1998; 9: 695-706Crossref PubMed Scopus (216) Google Scholar Two aspects of current production systems further compound the problem. First, vast amounts of input plasmids are used for AAV production and highly efficient transfection methods are employed,11Grieger J.C. Soltys S.M. Samulski R.J. 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: 287-297Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar such that replicating forms of AAV genomes are not required to be the predominant species in rAAV production system for high-yield rAAV production, which greatly increases the chances of packaging plasmid backbone into virions. Second, due to the necessity of downregulating rep78 during packaging,12Mouw M.B. Pintel D.J. Adeno-associated virus RNAs appear in a temporal order and their splicing is stimulated during coinfection with adenovirus.J. Virol. 2000; 74: 9878-9888Crossref PubMed Scopus (31) Google Scholar expression of the latter is repressed.13Li J. Samulski R.J. Xiao X. Role for highly regulated rep gene expression in adeno-associated virus vector production.J. Virol. 1997; 71: 5236-5243Crossref PubMed Google Scholar,14Xiao W. Chirmule N. Berta S.C. McCullough B. Gao G. Wilson J.M. Gene therapy vectors based on adeno-associated virus type 1.J. Virol. 1999; 73: 3994-4003Crossref PubMed Google Scholar Lower levels of rep78 protein in combination with other helper and cellular factors is presumably another contributing factor, such that newly replicating DNA is not the dominant substrate for the packaging machinery. Because current production systems are prone to packing non-vector plasmid sequences, the field should consider extending CpG depletion beyond the AAV vector genome into the plasmid backbone in order to further decrease immunogenicity of the vectors. Lax requirement of the D-element in rAAV packaging and inefficient replication of vector DNA are substantially affecting the composition of rAAV vectors used for human gene therapy, resulting in a high abundance of plasmid backbone contamination and increased presence of hypomethylated CpG sequences in rAAV genomes.2Wright J.F. Codon Modification and PAMPs in Clinical AAV Vectors: The Tortoise or the Hare?.Mol. Ther. 2020; 28: 701-703Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Increasing the size of the plasmid backbone was reported to significantly reduce, but not eliminate, reverse packaging.15Hauck B. Murphy S.L. Smith P.H. Qu G. Liu X. Zelenaia O. Mingozzi F. Sommer J.M. High K.A. Wright J.F. Undetectable transcription of cap in a clinical AAV vector: implications for preformed capsid in immune responses.Mol. Ther. 2009; 17: 144-152Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar Considering the less selective nature of the D-element in AAV encapsidation, it might be advantageous to adopt plasmid backboneless vector DNA for rAAV packaging. For instance, “doggie bone” DNA has been utilized for production of other biologicals such as lentiviral vectors. Finally, it has been proposed to develop production systems that incorporate methylation of CpG sequences, which might help drive high-level transgene expression.2Wright J.F. Codon Modification and PAMPs in Clinical AAV Vectors: The Tortoise or the Hare?.Mol. Ther. 2020; 28: 701-703Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Such an effort would certainly require an improved vector production system that eliminates direct packaging of unmethylated input vector sequences. If input DNA were fully methylated, limited DNA replication regulated by the D-element, might become an advantage since AAV DNA by itself is hypomethylated in the productive lytic infection. In conclusion, although D does not matter that much in rAAV packaging, it does matter immensely in vector performance and safety. Download .pdf (.72 MB) Help with pdf files Document S1. Figures S1 and S2