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. 2022 Dec 24;15(1):64.
doi: 10.3390/v15010064.

High-Level rAAV Vector Production by rAdV-Mediated Amplification of Small Amounts of Input Vector

Stefan Weger  1
Affiliations

High-Level rAAV Vector Production by rAdV-Mediated Amplification of Small Amounts of Input Vector

Stefan Weger. Viruses. .

Abstract

The successful application of recombinant adeno-associated virus (rAAV) vectors for long-term transgene expression in clinical studies requires scalable production methods with genetically stable components. Due to their simple production scheme and the high viral titers achievable, first generation recombinant adenoviruses (rAdV) have long been taken into consideration as suitable tools for simultaneously providing both the helper functions and the AAV rep and cap genes for rAAV packaging. So far, however, such rAdV-rep/cap vectors have been difficult to generate and often turned out to be genetically unstable. Through ablation of cis and trans inhibitory function in the AAV-2 genome we have succeeded in establishing separate and stable rAdVs for high-level AAV serotype 2 Rep and Cap expression. These allowed rAAV-2 production at high burst sizes by simple coinfection protocols after providing the AAV-ITR flanked transgene vector genome either as rAAV-2 particles at low input concentrations or in form of an additional rAdV. With characteristics such as the ease of producing the required components, the straightforward adaption to other transgenes and the possible extension to further serotypes or capsid variants, especially the rAdV-mediated rAAV amplification system presents a very promising candidate for up-scaling to clinical grade vector preparations.

Keywords: RIS-Ad; adeno-associated virus; rAAV packaging; recombinant adenovirus.

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Conflict of interest statement

S.W. is an inventor on European and International patent applications addressing the rAdV-mediated rAAV vector packaging technology described in this study.

Figures

Figure 1
Figure 1
Expression of AAV-2 Rep proteins by first generation recombinant adenoviruses (rAdV) after recoding of the 3′-inhibitory RIS-Ad sequence. (A) Genome organization of AAV-2 with a close-up of the RIS-Ad (Rep inhibitory sequence for adenoviral replication) in the 3′-part of the rep gene. The AAV-2 inverted terminal repeats (ITRs), the four Rep proteins Rep78, Rep68, Rep52, Rep40, the capsid proteins VP1 to VP3 and the AAP (assembly activating protein) are indicated by differentially colored boxes. The box “sRep“ in the lower part of the figure indicates recoding of AAV-2 nucleotides 1782 to 1916 sufficient for abolishing the inhibitory function of the RIS-Ad. Characteristic nucleotide positions are given above the boxes. (B) Schematic presentation of the transgene cassettes of the adenoviral shuttle plasmids tested. Formation of rAdV after transfection of the corresponding PacI-linearized pAdEasy plasmids into HEK293 cells is indicated by “+“ and “−“ signs. (C) Anti-Rep Western blot analysis after infection of HEK293 cells for 64 h with rAdV-MMTV-sRep (amplification round 5) and rAdV-M2-Tet-sRep (round 6) at the indicated MOIs (genomic particles/cell). Transfection of pDG plasmid (transfection efficiency about 70%) was used as positive control. (D) Anti-Rep Western blot analysis after infection of HEK293 cells with rAdV-MMTV-sRep as in (C) in the absence and presence of doxycycline. (E) Southern blot analysis for rAAV replicative DNA intermediates after transfection of HEK293 cells with pTR-UF5 and over-infection with rAdV-M2-Tet-sRep in the absence or presence of doxycycline at the indicated MOIs for 64 h. The positive control in lane 2 represents pTR-UF5/pDG co-transfected cells.
Figure 2
Figure 2
Improved rAdV-mediated expression of functional large Rep proteins by means of the constitutive HSV-TK promoter. (A) Genome organization of AAV-2 with a close-up of the RIS-Ad (Rep inhibitory sequence for adenoviral replication) in the 3′-part of the rep gene (for further details compare Figure 1A) and depiction of the large Rep protein Rep68 and the truncated Rep-Stop531 protein, which terminates immediately after the major AAV-2 splice donor site. (B) Anti-Rep Western blot analysis after co-infection of HEK293 cells with purified rAAV-GFP vector (MOI 100) and either rAdV-M2-Tet-sRep, rAd-HSV-TK-sRep68 or rAd-HSV-TK-sRep-Stop531, as indicated, for 64 h. Positive control are pTR-UF5/pDG co-transfected cells. (C) Same experiment as in (B) with extraction of viral DNA and Southern blot analysis with a probe for the CMV-GFP transgene cassette. Arrows indicate monomeric and dimeric replicative rAAV-GFP DNA intermediates. Representative data from a total of three experiments is shown.
Figure 3
Figure 3
Generation of rAdV for AAV-2 capsid protein expression. (A) Schematic presentation of the adenoviral shuttle plasmids. Formation of rAdV after transfection of the corresponding PacI-linearized pAdEasy plasmids into HEK293 cells is indicated by “+“ and “−“ signs. (B) Infection of HEK293 cells for 64 h with equal amounts of freeze–thaw supernatants from amplification rounds 3 to 8 of rAdV-HSV-TK-Cap amplification with subsequent anti-Cap western analysis of whole cell extracts. Arrows indicate positions of capsid proteins VP1 to VP3. pDG transfected HEK293 cells were used as positive control (C). As in (B) with rAdV-HSV-TK-Cap freeze–thaw supernatants from amplification rounds 6 (left lanes) and 5 (right lanes) at different MOIs for 64 h.
Figure 4
Figure 4
Setups for rAdV-mediated rAAV2-GFP vector packaging. The different experimental approaches for rAdV-mediated packaging of rAAV2-GFP vectors in HEK293 cells are illustrated: (I) infection of pTR-UF5 transfected cells with a combination of rAdV-M2-Tet-sRep or rAd-HSV-TK-sRep-Stop531 (generally denoted as rAdV-Rep for simplification) and rAdV-HSV-TK-Cap (denoted as rAdV-Cap), (II) triple rAdV infection with rAdV-Rep, rAdV-Cap and rAdV-ITR-GFP providing the AAV-2 ITR flanked CMV-GFP transgene cassette and (III) amplification of low input amounts of rAAV2-GFP by coinfection with rAdV-Rep and rAdV-Cap.
Figure 5
Figure 5
Optimization of rAdV-mediated rAAV2-GFP vector packaging. (A–D) Percentage of GFP positive cells obtained after transduction of 2 × 105 HeLa cells with small aliquots of freeze–thaw lysates from packaging experiments performed with 5 × 105 HEK293 cells in 6-well plates. For (A–C), 1/15 shares of the primary freeze–thaw lysates were used for transduction, while in (D) 1/30 shares were used. (A–C) correspond to the setups I to III illustrated in Figure 4 with rAdV-M2-Tet-sRep for expression of Rep proteins, while (D) corresponds to the setup III (rAAV-GFP amplification) with rAd-HSV-TK-sRep-Stop531 used for Rep expression. Input MOIs of the different vectors are indicated.
Figure 6
Figure 6
Characterization of affinity-purified rAAV2-GFP vector preparations generated with the help of recombinant adenoviruses. (A) Mean burst sizes in genomic particles per cell (vg/cell) for affinity-purified rAAV2-GFP vector preparations generated with different packaging approaches as indicated. The middle three bars involve rAdV-M2-Tet-sRep for expression of Rep proteins, while the right bar shows the preparations from the rAAV-GFP amplification protocol with rAd-HSV-TK-sRep-Stop531. Means were obtained from three preparations for the positive control (pTR-UF5/pDG co-transfection), the rAdV triple infection and the rAAV-GFP amplification mediated by the combination of rAd-HSV-TK-sRep-Stop531 and rAdV-HSV-TK-Cap. For the remaining setups, two preparations were averaged. Standard deviations are indicated by the corresponding error bars. (B) Percentage of GFP positive cells after transduction of HeLa cells with 5 different MOIs (expressed in vg/cell) of affinity purified rAAV2-GFP preparations from different setups. Means and standard deviations shown by errors bars were obtained from experiments with three positive control rAAV preparations and two preparations each for the remaining setups described in detail in (A) and in the text. (C,E) Silver staining of SDS PAGE gels loaded with different amounts of genomic particles, as indicated, from selected purified rAAV2-GFP vector preparations. For (C) the respective preparations with the highest burst size for the four different packaging methods presented in the first four bars of part A were analyzed, while for (E) the two preparations with the highest burst sizes for the positive control and the rAd-HSV-TK-sRep-Stop531/rAdV-HSV-TK-Cap-mediated rAAV amplification were analyzed. (D,F) Western blot analysis of capsid protein content with different amounts of genomic particles from the same rAAV2-GFP vector preparations as in (C,E).
Figure 7
Figure 7
Genomic stability of rAdV used in rAAV packaging. (A,B) rAdV-M2-Tet-sRep and rAdV-HSV-TK-Cap were amplified in HEK293 cells over 15 round as described in the methods section. (A) Determination of genomic rAdV titers. (B) Analysis of the genomic integrity by PCR-amplification with oligonucleotides binding at the very ends of the respective transgene cassette. The pAdEasy plasmids used for generation of the corresponding rAdVs were used as positive controls. (C,D) Analysis of the transgene cassette in supernatants from different amplification rounds of (C) rAdV-HSV-TK-sRep68 and (D) rAdV-HSV-TK-sRep-Stop531. The oligonucleotides used for PCR amplification bind within the adenoviral sequences flanking the transgene cassette in the pAdEasy plasmids, which were also used as positive controls.
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This research received funding from the SPARK/BIH program of the Berlin Institute of Health (BIH).

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