Improved splicing of adeno-associated viral (AAV) capsid protein-supplying pre-mRNAs leads to increased recombinant AAV vector production

Abstract

Adeno-associated viral (AAV) capsid proteins, thought to be a rate-limiting step in the production of recombinant AAV (rAAV), are translated from spliced mRNAs. Improvement of the native AAV nonconsensus donor sequence increases splicing yet leaves the relative levels of VP1- and VP2/3-encoding mRNAs unchanged, and thus provides a means to increase delivery of correct ratios of AAV capsid proteins. This effect is independent of the AAV serotype used, and occurs whether the rep and cap genes supplied in trans are on the same or separate expression vectors. In the split-vector system, replacement of the more traditionally used cytomegalovirus promoter with that of the AAV5 P41 promoter allowed for even greater levels of splicing, and together with an improved intron donor, led to a 10- to 15-fold increase in the levels of splicing, rAAV production, and transduction compared with levels achieved by traditional cotransfection methods. Thus, the enhancement of splicing presents a useful method to enhance rAAV production via transient transfection.

FIG. 1.

Improvement of the nonconsensus AAV donor significantly enhanced overall pre-mRNA splicing, capsid production, and rAAV production. Left: Representative RNase protection assay of AAV1 and AAV2 RepCap plasmids (AV1RC and AV2RC, respectively) with wild-type, “better” (bD), or consensus donor (cD) mutations in the presence of pHelper. Unsp, Unspliced; Sp, Spliced. Top right: Quantification of the relative spliced-to-unspliced ratios of capsid-encoding pre-mRNA. Data are from at least three experiments and show standard deviations. The position of the “RP” RNase protection probe is indicated. Bottom right: Relative levels of rAAV production observed for AAV1, AAV2, and AAV8 with the “better” and consensus donor mutants in relation to levels obtained with the wild-type donors (set to a value of 1.00). Average titers of DNase-resistant rAAV virions per 100-mm dish (with standard deviations in parentheses) were as follows: AV1RC, 1.25 × 10¹⁰ (±4.87 × 10⁹); AV1bD, 4.78 × 10¹⁰ (±2.16 × 10¹⁰); AV1cD, 1.08 × 10¹⁰ (±2.75 × 10⁹); AV2RC, 3.43 × 10¹⁰ (±1.33 × 10¹⁰); AV2bD, 1.15 × 10¹¹ (±4.10 × 10¹⁰); AV2cD, 3.04 × 10¹⁰ (±1.34 × 10¹⁰); AV8RC, 6.97 × 10¹⁰ (±2.01 × 10¹⁰); AV8bD, 2.78 × 10¹¹ (±9.07 × 10¹⁰); AV8cD, 4.45 × 10¹⁰ (±1.72 × 10¹⁰).

FIG. 2.

Use of a split Rep/Cap AAV helper system overcame the negative effects of the consensus donor, allowing for even greater levels of rAAV production. Left: Representative RNase protection assay of AAV2 CMV-driven capsid-encoding pre-mRNAs in the absence or presence of pHelper. Middle: Quantification of the relative spliced-to-unspliced ratios of capsid-encoding pre-mRNA. Data are from at least three experiments and show standard deviations. The position of the “RP” RNase protection probe is indicated. Right: Relative levels of rAAV production observed in AAV2, AAV6, and AAV8 with the “better” and consensus donor mutants in relation to levels obtained with the wild-type donors (set to a value of 1.00). Average titers of DNase-resistant rAAV virions per 100-mm dish (with standard deviations in parentheses) were as follows: CMV-Cap2, 1.95 × 10¹⁰ (±6.75 × 10⁹); CMV-Cap2bD, 7.47 × 10¹⁰ (±1.78 × 10¹⁰); CMV-Cap2cD, 1.15 × 10¹¹ (±4.82 × 10¹⁰); CMV-Cap6, 9.24 × 10⁹ (±6.84 × 10⁸); CMV-Cap6bD, 3.75 × 10¹⁰ (±2.41 × 10⁹); CMV-Cap6cD, 5.45 × 10¹⁰ (±3.27 × 10⁹); CMV-Cap8, 2.45 × 10¹⁰ (±7.27 × 10⁹); CMV-Cap8bD, 8.59 × 10¹⁰ (±1.56 × 10¹⁰); CMV-Cap8cD, 1.25 × 10¹¹ (±3.06 × 10¹⁰).

FIG. 3.

The AAV5 P41 promoter allows for more efficient splicing of capsid-encoding AAV pre-mRNA than the CMV promoter, and together with an improved donor, led to the generation of high levels of rAAV. Left: RNase protection assay of CMV- and P41-driven AAV2 capsid-encoding pre-mRNAs in the presence of pHelper. Middle: Quantification of the relative spliced-to-unspliced ratios of capsid-encoding pre-mRNA. Data are from at least three experiments and show standard deviations. The position of the “RP” RNase protection probe is indicated. Right: Relative levels of rAAV production observed in AAV2 and AAV8 with the “better” and consensus donor mutants, in addition to the P41 wild-type and consensus donor mutants, in relation to levels obtained with the CMV-driven wild-type donor capsid-encoding construct (set to a value of 1.00). Titers of rAAVs generated with the CMV-driven wild-type donor construct typically ranged from approximately 9.5 × 10⁹ to 2.0 × 10¹¹ packaged genomes per 100-mm dish. Average titers of DNase-resistant rAAV virions per 100-mm dish (with standard deviations in parentheses) were as follows: CMV-Cap2, 8.59 × 10¹⁰ (±3.41 × 10¹⁰); CMV-Cap2bD, 3.33 × 10¹¹ (±1.28 × 10¹¹); CMV-Cap2cD, 5.02 × 10¹¹ (±1.72 × 10¹¹); P41-Cap2, 5.05 × 10¹¹ (±2.34 × 10¹¹); P41-Cap2cD, 1.08 × 10¹² (±4.40 × 10¹¹); CMV-Cap8, 2.77 × 10¹¹ (±1.57 × 10¹¹); CMV-Cap8bD, 1.10 × 10¹² (±6.83 × 10¹¹); CMV-Cap8cD, 2.18 × 10¹² (±1.53 × 10¹²); P41-Cap8, 1.31 × 10¹² (±5.89 × 10¹¹); P41-Cap8cD, 4.44 × 10¹² (±3.18 × 10¹²).

FIG. 4.

Levels of transduction-capable rAAV correlate well with the increased levels of packaged genomes. Shown are the results of a transduction assay of HeLa cells, plated on 24-well dishes 24 hr before infection with 10-fold serial dilutions of rAAV8 (5 × 10⁷ packaged genomes per well). GFP-positive cells were counted 48 hr postinfection. Top left: Phase-contrast microscopy. Bottom left: GFP-positive cells. Right: The number of packaged genomes per well was divided by the approximate number of GFP-positive transduced cells to obtain the efficiency of infection. Values indicated are expressed as a ratio of packaged genomes per transducing unit. The ssDNA/TU ratio for all rAAVs tested in the absence of Ad ranged from approximately 1400 to 2000. Data are from three experiments and show standard deviations.