Existence of transient functional double-stranded DNA intermediates during recombinant AAV transduction

Abstract

Previous studies have documented that 0.1 approximately 1% of input recombinant adeno-associated virus (rAAV) vectors could be stabilized and lead to transgene expression. To characterize the steps involving massive AAV DNA loss, we designed an"AAV footprinting" strategy that can track newly formed AAV dsDNA genomes. This strategy is based on an ROSA26R mouse model or cell line that carries a lacZ gene flanked by two loxP sites. When it is transduced by a rAAV vector carrying the Cre recombinase, the lacZ gene can be activated and remain active even when rAAV genomes are later lost. By using this sensitive AAV footprinting technique, we confirmed the existence of transient AAV dsDNA that went undetected by conventional DNA methods. Although these dsDNA intermediates could be efficiently formed in almost every cell and were competent for mRNA transcription and protein synthesis in vivo, they got lost continuously. Only a small fraction was eventually stabilized for sustained gene expression. Although both rAAV2 and rAAV8 can potentially have similar levels of dsDNA formation, AAV8 dsDNA was formed much faster than that of AAV2, which explains why rAAV8 is more efficient than rAAV2 in transducing the liver. Collectively, our studies suggested that rather than receptor binding, viral entry, and ssDNA to dsDNA conversion, the instability of newly formed AAV dsDNA was the primary contributing factor for the low rAAV transduction efficacy. The uncoating step significantly influenced the stability of AAV transient dsDNA. The identification of transient AAV dsDNA provided a new pathway for improving rAAV transduction.

Fig. 1.

AAV genome footprinting strategy. (A) Schematic representation of the AAV-CB-ALP and AAV-CB-cre vectors used in this work. The locations of restriction enzymes and probe for Southern blotting are marked. PolyA, the human β-globin gene polyadenylation signal; s, stuffer sequence without function; B, BglII; E, EcoRI. (B) Schematic of lacZ activation by Cre in C2lacZ cells and ROSA26R (Gtrosa26tm1Sor) mice. (C) Strategy of AAV dsDNA footprinting. Key steps are shown: 1, rAAV entry; 2, uncoating, dsDNA synthesis, lacZ gene activation; 3, dsDNA get lost; 4, dsDNA stabilization. X stands for the stop sequence in the lacZ gene expression cassette.

Fig. 2.

rAAV transduction in C2lacZ cells. C2lacZ cells were infected with AAV-CB-ALP and AAV-CB-cre (1:1) at a multiplicity of infection of 10,000. The expression of ALP or LacZ was analyzed at specified time points as indicated. (A) ALP or LacZ expression of C2lacZ cells infected with AAV2 vectors. (Upper) Nondividing C2lacZ cells. (Lower) Dividing C2lacZ cells. (Scale bar: 100 μm.) (B–E) Quantification of ALP- or LacZ-positive cells after AAV2 or AAV8 transduction of C2lacZ cells. The y axis stands for the percentage of cells positive for ALP or LacZ staining. Error bars indicate the SD values.

Fig. 3.

AAV dsDNA footprinting in the liver. AAV2 or AAV8 carrying ALP or cre was administered i.v. to ROSA26R mice at a dose of 1 × 10¹¹ vector genomes. (A) Staining of ALP or LacZ expression in the liver section of mice receiving AAV2-based vectors at various time points after vector administration. Neg represents a negative control before vectors were administered. (Scale bar: 100 μm.) (B) Staining of ALP or LacZ expression in the liver section of mice receiving AAV8-based vectors at various time points after vector administration. (C) Southern blot analysis of rAAV genome after vector administration. Total cellular mouse liver DNA was extracted from liver samples of each time point. Twenty micrograms of BglII-, MfeI-, and EcoRV-digested genomic DNA was separated on 0.8% agarose gel. Hybridization was performed by using CB promoter probe recovered by EcoRI. Genome copy number standards are shown in the figure. Fragment size corresponding to the ALP or cre vector is also indicated. ss represents the ssDNA from the incoming vector.

Fig. 4.

Instability of scAAV genomes. Self-complementary AAV2 vectors carrying TTR-ALP or TTR-cre (1:1) were administered i.v. to the ROSA26R mice at a dose of 1 × 10¹¹ vector genomes. Liver were harvested at various time points after vector administration, and the ALP and lacZ gene expression was analyzed by histochemical staining. Neg represents a negative control before vectors were administered. (Scale bar: 100 μm.)

Fig. 5.

rAAV genomes remain mostly as episomes. ROSA26R mice were injected with AAV-CB-ALP and AAV-CB-cre (1:1) at a dose of 1 × 10¹¹ vector genomes per animal through the tail vein. After 6 weeks, half of the mice (n = 3) underwent partial hepatectomy (PH). All animals were killed another 6 weeks later. ALP or LacZ staining of the liver sections before and after PH is shown. Control mice receiving saline were subjected to the same PH procedure. (Scale bar: 100 μm.)

Fig. 6.

Theoretical model for transient AAV dsDNA in rAAV transduction life cycle. (A) Transient dsDNA status during AAV2 transduction. (B) Transient dsDNA during AAV8 transduction. The difference between AAV2 and AAV8 in uncoating is noted in the figure as “slow” and “fast.” The traditional rate-limiting step is also noted in the figure. **, all of the steps that may involve degradation. The vector DNA illustrated by dotted line represents lost AAV genomes.