Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors

Abstract

Different approaches are used in the production of recombinant adeno-associated virus (rAAV). The two leading approaches are transiently transfected human HEK293 cells and live baculovirus infection of Spodoptera frugiperda (Sf9) insect cells. Unexplained differences in vector performance have been seen clinically and preclinically. Thus, we performed a controlled comparative production analysis varying only the host cell species but maintaining all other parameters. We characterized differences with multiple analytical approaches: proteomic profiling by mass spectrometry, isoelectric focusing, cryo-EM (transmission electron cryomicroscopy), denaturation assays, genomic and epigenomic sequencing of packaged genomes, human cytokine profiling, and functional transduction assessments in vitro and in vivo, including in humanized liver mice. Using these approaches, we have made two major discoveries: (1) rAAV capsids have post-translational modifications (PTMs), including glycosylation, acetylation, phosphorylation, and methylation, and these differ between platforms; and (2) rAAV genomes are methylated during production, and these are also differentially deposited between platforms. Our data show that host cell protein impurities differ between platforms and can have their own PTMs, including potentially immunogenic N-linked glycans. Human-produced rAAVs are more potent than baculovirus-Sf9 vectors in various cell types in vitro (p < 0.05-0.0001), in various mouse tissues in vivo (p < 0.03-0.0001), and in human liver in vivo (p < 0.005). These differences may have clinical implications for rAAV receptor binding, trafficking, expression kinetics, expression durability, vector immunogenicity, as well as cost considerations.

Keywords: AAV; PTM; adeno-associated virus; baculovirus-Sf9; capsid; human; mass spectrometry; methylation; post-translational modification; potency.

Graphical abstract

Figure 1

rAAV Capsids Manufactured with the Human and Baculovirus-Sf9 Platforms are Post-Translationally Modified and Exhibit Differential PTM Profiles (A) PTM identities and residue positions along the length of the rAAV8 polypeptide from the N to C terminus in the baculovirus (baculo-)Sf9 vector. PTMs are colored by type (acetylation, green; methylation, blue; phosphorylation, cyan; deamidation, orange; O-GlcNAcylation, magenta). Residues above the sequence are externally facing on the capsid. Residues below are lumenal or buried. Residues within the gray box from 1 to 220 represent the disordered region of AAV8 yet to be crystallized. The two regions for LamR binding (491–547 and 593–623) are highlighted in yellow boxes. (B) Cumulative capsid PTMs observed from all baculo-Sf9 rAAV8 lots, purified from both cell lysates and media. Same color code as in (A). (C) Same as (A) but with human-produced rAAV8. (D) Same as (B) but with human rAAV8. (E) Shared and unique capsid PTMs for rAAV8 produced in the baculo-Sf9 (yellow) and human (purple) platforms. Same color code as in (A). Excluded are deamidation degradation marks which are universal. (F) Negative staining and TEM imaging of baculo-Sf9 rAAV8 cell-purified vector. White arrow indicates full capsid; red arrow indicates empty capsid; for reference for (F)–(I) (percent full capsids noted on left). Original magnification, ×20,000. (G) Same as (F) but media-purified vector. (H) Same as (F) but with human rAAV8 cell-purified vector. (I) Same as (H) but with media-purified vector. (J) Silver stain of capsid VP species present in vector lots from (F)–(I). (K) 2D gel images from human-produced rAAV8 from pH 3 to pH 10. VP1 (87 kDa), VP2 (72 kDa), and VP3 (62 kDa) bands are indicated with black arrowheads. (L) 2D gel images from baculo-Sf9-produced rAAV8. (M) Thermal capsid melt curves for rAAV8 vectors shown from 50°C to 90°C; full melt curves from 30°C to 95°C are in Figure S7A. T_m initiation, dashed black line; final T_m, dashed gray line.

Figure 2

Human and Baculovirus-Sf9 Production Platforms Produce rAAV Capsids with Similar Structures by Cryo-EM (A) Overlay of an individual VP1 chain from human (red-orange) and baculovirus (baculo-)Sf9 (cyan) full rAAV8 capsids highlighting an absence of macro-level differences. Key structural landmarks are shown. (B) Magnified overlay of the LamR binding region from a human (red-orange electron density caging) and baculo-Sf9 (cyan) full rAAV8 capsid highlighting residues capable of post-translational modification as determined by LC-MS/MS. (C) Magnified overlay depicting potential side-chain level structural differences in phenyl ring orientations between human and baculo-Sf9 rAAV8. (D) Magnified overlay of a single human and baculo-Sf9 rAAV8 capsid cylinder loop depicting minor potential side-chain level differences.

Figure 3

Packaged rAAV Genomes Are Methylated during Production and Differentially Methylated Based on the Manufacturing Platform The five tracks of the Circos plot from the outside-in represent the following. First, a schematic of the ssAAV8 genome sequenced with key features labeled is shown (inverted terminal repeat [ITR], orange; intervening sequences, gray; EF1α promoter, red; firefly luciferase expression cassette, blue; woodchuck hepatitis virus posttranscriptional regulatory element [WPRE], plum; human growth hormone polyadenylation signal [hGHpA], magenta). Second, in blue, the methylation ratios for all detected sites for baculovirus-Sf9-produced rAAV8 are shown; the plotting represents a minimum of 0 and a maximum of 1 in 0.1 increments. Third, in red, the methylation ratios for all detected sites for human-produced rAAV are shown; scale same as previous. Fourth, in blue, the total numbers of reads at detected sites for baculovirus-Sf9 produced rAAV8 are shown; in log₂ scale, from a minimum of 0 (total reads of 1) to a maximum of 11 (total reads of 2¹¹), in 0.1 increments. Fifth, in red, the total numbers of reads at detected sites for human-produced rAAV are shown; scale same as previous. The three sites with statistically significant differential methylation are highlighted in green (C:1208, C:2970, C:3779).

Figure 4

rAAV Produced with the Human Platform Is More Potent In Vitro and in Skeletal Muscle In Vivo following Intramuscular Administration (A) In vitro functional transduction assays in immortalized human HEK293T and Huh7 cells, primary human fibroblasts, primary human iPSCs, and immortalized mouse C2C12 myoblasts transduced with ssAAV1-EF1α-FLuc. Human-produced rAAV1 was significantly more potent than similar baculovirus (baculo-)Sf9 vector in all cases. (B) HEK293T cells were transduced with varying ratios of full/empty human-produced ssAAV1-EF1α-FLuc with the total capsid content kept constant at an MOI of 30,000, while the ratio of full/empty varied. FLuc assays were performed on cell lysates 3 days post-transduction and normalized to mock-transduced wells. Each green dot represents one replicate; mean ± SD is shown. (C) Same as (A) except using baculo-Sf9 rAAV1. (D) To further assess the impact of insect HCP impurities, HEK293T cells were transduced with a fixed MOI of 30,000 of full baculo-produced ssAAV1-EF1α-FLuc and spiked with an additional 10% or 100% of empty baculo-produced or empty human-produced vector. FLuc assays were performed on cell lysates 3 days post-transduction and normalized to mock-transduced wells. Each dot represents one replicate; mean ± SD is shown. (E) In vivo time course functional transduction assays comparing human and baculovirus-Sf9 ssAAV1-EF1α-FLuc after i.m. administration (5e10 vg/mouse) in age-matched siblings. Mean radiance (photons/s/cm²/sr) displayed with all mice imaged on their ventral side on the same shared scale. (F) Same as (E) but with ssAAV8-EF1α-FLuc; same shared scale as (E). (G) Quantification of rAAV1 FLuc radiance from (E). Each symbol represents mean signal (±SD) from three mice. (H) Same as (G) but with rAAV8. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.

Figure 5

Human-Produced rAAV Demonstrated Significantly More Functional Liver Transduction In Vivo following Intravenous Administration and Functional Transduction Is Sexually Dimorphic with rAAV from Both Platforms (A) In vivo time course functional transduction assays comparing human and baculovirus (baculo-)Sf9-produced ssAAV1-EF1α-FLuc after i.v. tail vein administration (5e10 vg/mouse) in age-matched siblings. Mean radiance (photons/s/cm²/sr) displayed with all mice imaged on their ventral side on the same shared scale. (B) Same as (A) but with rAAV8; same shared scale as (A). (C) Quantification of (A). Human rAAV1 is more potent than baculo-Sf9 rAAV1 (p < 0.005–0.0001 at week 4), and males have higher functional liver transduction than do females (p < 0.04–0.0001). (D) Quantification of (B). Human rAAV8 is also more potent than baculo-Sf9 rAAV8 (p < 0.03–0.0001), and males again have higher functional liver transduction than do females (p < 0.009–0.0001). Detailed statistics are shown in Table S23.

Figure 6

rAAV Produced Using the Human Manufacturing Platform Has Significantly More Functional Human Liver Transduction In Vivo (A) Schematic illustrating the production of humanized liver mice used for assessing comparative functional human liver transduction in vivo with rAAV expressing Firefly luciferase (FLuc). (B) In vivo time course functional transduction assays comparing human and baculovirus (baculo-)Sf9-produced ssAAV1-EF1α-FLuc after i.v. tail vein administration (5e10 vg/mouse) in age-matched humanized liver mice. Mean radiance (photons/s/cm²/sr) is displayed with all mice imaged on their ventral side on the same shared scale. (C) Quantification of rAAV1 FLuc radiance in male human liver. Each symbol indicates mean signal (±SD) from four humanized mice. (D) Quantification of rAAV1 FLuc radiance in female human liver. Each symbol indicates mean signal (±SD) from three humanized mice. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.