Homologous recombination is required for AAV-mediated gene targeting

Abstract

High frequencies of gene targeting can be achieved by infection of mammalian cells with recombinant adeno-associated virus (rAAV) vectors [D. W. Russell and R. K. Hirata (1998) Nature Genet., 18, 325-330; D. W. Russell and R. K. Hirata (2000) J. Virol., 74, 4612-4620; R. Hirata et al. (2002) Nat. Biotechnol., 20, 735-738], but the mechanism of targeting is unclear and random integration often occurs in parallel. We assessed the role of specific DNA repair and recombination pathways in rAAV gene targeting by measuring correction of a mutated enhanced green fluorescent protein (EGFP) gene in cells where homologous recombination (HR) or non-homologous end-joining (NHEJ) had been suppressed by RNAi. EGFP-negative cells were transduced with rAAV vectors carrying a different inactivating deletion in the EGFP, and in parallel with rAAV vectors carrying red fluorescent protein (RFP). Expression of RFP accounted for viral transduction efficiency and long-term random integration. Approximately 0.02% of the infected GFP-negative cells were stably converted to GFP positive cells. Silencing of the essential NHEJ component DNA-PK had no significant effect on the frequency of targeting at any time point examined. Silencing of the SNF2/SWI2 family members RAD54L or RAD54B, which are important for HR, reduced the rate of stable rAAV gene targeting approximately 5-fold. Further, partial silencing of the Rad51 paralogue XRCC3 completely abolished stable long-term EGFP expression. These results show that rAAV gene targeting requires the Rad51/Rad54 pathway of HR.

Figure 1

Schematic representation of the vectors used for gene targeting. (A) Plasmid pEGFPΔ32 containing the mutant target (thick white arrow) expressed under human CMV promoter was integrated in the genome of MO59K cells. Thirty-two base pair deletion at position 198 (5′-ctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaa-3′) was replaced by an in-frame stop-codon supplied within a unique SpeI restriction site (5′-CACTAGTTGAAGCa-3′). Resistance to hygromycin was provided by a hygromycin phosphotransferase (Hygro) expressed from an SV40 promoter. (B) Schematic of rAAV used as repair substrates in gene targeting experiments. All viral vectors consisted of single-stranded DNA enclosed by native AAV subtype 2 inverted repeats. Both substrates (white arrows) lacked 14 bp from the 5′ end of the EGFP gene including the ATG codon (Δ14EGFP). A short and a long fragment from the P.pastoris alcohol oxidase termination sequence (Pp AOX TT and TT) replaced the CMV and the CAAT+TATA signals of the CBA/rabbit β-globin promoter (CBA/β-globin). The rAAV-NeoX contained additional homology to the genomic target (thick black line). The recombinant viruses also contained a neomycin phosphotransferase gene (Neo) expressed under a HSV-TK promoter. (C) Diagram of the vectors used to test cell infectivity. rAAV-RFPX and rAAV-RFP were identical to rAAV-NeoX and rAAV-Neo, respectively, except for the replacement of the neomycin phosphotransferase expression cassete with a RFP expression.

Figure 2

Evidence of accurate gene correction. (A) Optimization of rAAV infection. A total of 3 × 10⁵ cells of MO59KΔ32 polyclonal population (diagonal stripes), clone MO59KΔ32 A (white columns) and clone MO59KΔ32 B (vertical stripes) were infected with rAAV-Neo (upper chart) or rAAV-NeoX (lower chart) at the indicated MOI. The virus contaning medium was removed 24 h later. EGFP positive cells were counted by flow cytometry on day 2. (B) Southern analysis of SpeI digested genomic DNA with a probe consisting of full-length EGFP. Wild-type MO59K cells (lane1) served as a negative control. DNA from uninfected MO59KΔ32 B clone was loaded in lane 2. Lanes 3 to 10 contain genomic DNA from individual EGFP positive clones derived from polyclonal rAAV-NeoX transduced population. DNA ladder sizes are shown to the right. A schematic bellow illustrates the variety of fragments that may arise from head-to-tail (H-T ^*), head-to-head (H-H) or tail-to-tail (T-T) dimers of the targeting substrate integrated in the same genomic locus of MO59KΔ32 B. The band arising from digestion of the EGFPΔ32 mutant with SpeI is marked by an arrowhead (‘+SpeI’). The fragment containing repaired EGFP that lacks SpeI restriction site is marked with an arrow (‘−Spe I’). (C) PCR of genomic DNA amplifying 320 bp of corrected EGFP and 288 bp of mutant EGFPΔ32 fragment. Lane 3 contains the products from rAAV-NeoX transduced polyclonal population. Lanes 4–6 represent product amplified from single clones derived from the same polyclonal population. DNA from wild-type MO59K cells (lane 1) and EGFP expressing cell line (lane 7) were used as a negative and positive control, respectively.

Figure 3

DNA-PKcs is not required for stable rAAV-mediated targeting. (A) Immunoblot analysis of specific RNAi-mediated silencing of DNA-PKcs in MO59KΔ32 cells. Lane sh6684 and sh6684* depict the downregulation of DNA-PKcs expression pre- and post-infection, respectively. Sh11838 represents inefficient downregulation of expression at the respective position of the mRNA. Nuclear extract from DNA-PKcs mutant MO59J cell line (derived from MO59K) was used as a negative control. The relative level of residual protein expression with respect to MO59KΔ32 cells is shown at the bottom of each lane. (B) Time-course analysis of rAAV infection. 3 × 10⁵ cells were infected with rAAV-RFPX and the virus-containing medium removed 24 h later. Cells were passaged and the percentage of RFP positive cells determined by FACS in fraction of the cells. (C and D) Time-course analysis of rAAV gene targeting. 3 × 10⁵ cells were infected with 50 000 MOI of rAAV-NeoX. The virus was removed 24 h later and half of the cells assayed by FACS for GFP expression. The rest of the cells were re-plated and subsequently used for analysis every other day.

Figure 4

Rapid removal of rAAV episomes results in reduced targeting rates. (A) FACS analysis of gene targeting in MO59KΔ32 cells at the indicated days after transduction with rAAV-NeoX. GFP positive (%) gates define the number of cells with low and high fluorescence intensity of GFP. Targeted events occurring on the virus DNA are represented by cells exhibiting low intensity of fluorescence (full line) and repair of the genomic target is measured by cells exhibiting strong fluorescence (interrupted line). (B) FACS analysis of 293T cells transfected with low molecular weight DNA isolated from rAAV-RFPX infected cells. (C) FACS analysis of 293T cells transfected with low molecular weight DNA isolated from rAAV-NeoX infected cells. (D) and (E) Slot-blot analysis of of rAAV DNA isolated from total cell lysates at day 7, day 15 and day 21 post-infection. To quantify rAAV-NeoX DNA (D) a reference plasmid pTR-NeoX was used for standard curve (STD) and the blot was hybridized with Neo probe. (E) Slot-blot membrane re-probed with a GAPDH probe to quantify relative cell numbers.

Figure 5

Gene targeting frequency of rAAV corellates with RAD54B expression levels. (A) Immunoblot analysis of RAD54B-specific silencing. MO59KΔ32 designates the founder EGFPΔ32 expressing cell line. sh1409R54B, sh1512R54B, sh475R54B and sh1511R54B indicate the start position of the shRNA oligo on the Rad54B mRNA. The relative level of residual protein expression with respect to the founder cells is shown at the bottom of each lane. (B) Monitoring of infection efficiency. Cells were transduced with rAAV-RFPX and the number of RFP positive cells was counted by flow cytometry in aliquots from passaged cells. (C and D) Time-course analysis of rAAV gene targeting. (D) shows a scaled-up version of the later time-points from (C).

Figure 6

Strong inhibition of RAD54L expression leads to reduced rAAV targeting frequency. (A) Immunoblot analysis of Rad54L-specific silencing. MO59KΔ32 B+Eco designates the founder MO59KΔ32 B clone expressing Ecotropic receptor. sh1472R54L, sh2281R54L and sh1396R54L indicate the start position of the retrovirally expressed shRNA oligo on the RAD54L mRNA. SMC1α and β-actin were used as loading controls and nuclear extract from mouse ES Rad54± cells was used as a RAD54 size-reference. (B) Time-course analysis of rAAV infection. MO59KΔ32 B+Eco cells and RAD54L silenced polyclonal populations were transduced with rAAV-RFPX to test infectivity. The fraction of cells expressing RFP was determined every other day in an aliquot of passaged cells. (C) Gene targeting of MO59KΔ32 B+Eco and RAD54L silenced cells. 5 × 10⁵ cells were transduced with 50 000 MOI of rAAV-NeoX and GFP positive cells detected by flow cytometry on alternate days starting 24 h after infection.

Figure 7

Suppression of XRCC3 abolishes long-term gene correction. (A) Northern analysis of XRCC3 silencing. MO59KΔ32 designates the founder cell line and sh914, sh2059, sh362 and sh367 indicate the type of shRNA used to downregulate XRCC3 expression in the respective puromycin resistant population the total RNA was isolated from. GAPDH was used as an internal control to normalize for loading. (B) Comparison of infectivity of silenced and non-silenced cells. MO59KΔ32 B and their sh367XRCC3 derivative were transduced with rAAV-RFPX. As a measure of infection efficiency the level of RFP expression in the total cell population was monitored on alternate days. (C) Gene targeting in non-silenced and XRCC3 silenced cells. MO59KΔ32 B and sh367XRCC3 cells were transduced with 50 000 MOI of rAAV-NeoX. The GFP positive cells were detected by flow cytometry every other day for 15 days after infection. (D) Enlarged version of (C) spanning day 7 to day 15 after infection, which enables viewing the number of stable targeting events in detail.

Figure 8

Possible mechanism of gene targeting by rAAV. After the viral capsid has been removed RPA may bind to the single-stranded AAV genome and support initiation of HR. The BRCA2-RAD51 complex, assisted by RAD51 paralogs, such as XRCC3, replaces RPA with RAD51. The nucleoprotein filament finds and pairs with the homologous chromosomal sequence via unwinding/supercoiling facilitated by RAD54. Non-crossover gene conversion by repair DNA synthesis can occur either on the virus DNA (as shown here) or on the chromosomal target. Alternatively, resolution of vector-chromosomal DNA intermediates (arrows mark the two possible options) results in introduction of targeted modification in the homologous chromosome by crossing over and exchange of sequences. Only two of four possible outcomes are shown. The images may not represent the actual sequence of events.