A comparison of AAV-vector production methods for gene therapy and preclinical assessment

Abstract

Adeno Associated Virus (AAV)-mediated gene expression in the brain is widely applied in the preclinical setting to investigate the therapeutic potential of specific molecular targets, characterize various cellular functions, and model central nervous system (CNS) diseases. In therapeutic applications in the clinical setting, gene therapy offers several advantages over traditional pharmacological based therapies, including the ability to directly manipulate disease mechanisms, selectively target disease-afflicted regions, and achieve long-term therapeutic protein expression in the absence of repeated administration of pharmacological agents. Next to the gold-standard iodixanol-based AAV vector production, we recently published a protocol for AAV production based on chloroform-precipitation, which allows for fast in-house production of small quantities of AAV vector without the need for specialized equipment. To validate our recent protocol, we present here a direct side-by-side comparison between vectors produced with either method in a series of in vitro and in vivo assays with a focus on transgene expression, cell loss, and neuroinflammatory responses in the brain. We do not find differences in transduction efficiency nor in any other parameter in our in vivo and in vitro panel of assessment. These results suggest that our novel protocol enables most standardly equipped laboratories to produce small batches of high quality and high titer AAV vectors for their experimental needs.

Figure 1

Experimental outline: (A) Description of the seven experimental groups. (B) Schematic of the self-complimentary (sc) viral genome expressing GFP under the CMV promoter injected into the midbrain of rats. GFP green fluorescent protein, Iod iodixanol, Chl chloroform, SNpc Substantia nigra pars compacta, Str striatum, ITR inverted terminal repeat, CMV cytomegalovirus promoter, pA polyadenylation site.

Figure 2

Total protein levels and capsid assessment. (A) Coomassie blue gel displaying protein-weight distribution of the two AAV-GFP vectors produced with the Iodixanol-gradient and the Chloroform-precipitation method compared to a BSA control. The bands expected for the three capsid proteins are highlighted with white arrows and the molecular weight for BSA is highlighted with a blue arrow. (B) Western-blot for the three capsid proteins VP1, VP2 and VP3 for the four groups containing viral capsids. Figures A and B are cropped to highlight the relevant information. Full sized gels are presented in Supplementary Fig. S1. (C) Bar graph displaying the number of full and empty capsid particles counted. (D) TEM images displaying empty (red arrows) and full capsids (green arrows) for either production method.

Figure 3

In vitro infectivity assay. Results from the in vitro transduction of HeLa cells for decreasing gc AAV-GFP vector produced from the Iod-GFP (A–j) and Chl-GFP (K–t) production methods. Representative low magnification images from the respective groups (Iod-GFP: A–j, Chl-GFP: K–t) clearly display the decrease in fluorescence intensity. High magnification confocal microscopy images from the centre of the wells (a–j and k–t) indicate that there are a few cells expressing GFP even at relatively low gc-titers. (U) Quantifications for actual cell numbers co-expressing DAPI and GFP. Each condition was repeated in triplicates. Note that a conservative threshold was chosen hence underestimating the true transduction rates.

Figure 4

In vivo comparison of production methods on transgene expression and inflammation. Overview of a coronal 1:12 series of the brains labelled for GFP for the two main experimental groups (A-Iod-GFP; B-Chl-GFP) as well for the PBS-injected control group (C). Note that overviews for the four remaining control groups are supplied in the Supplementary Fig. S2). (D–G) High power confocal microscopy images displaying the strong expression of GFP in the terminal fibres in the striatum (D,F) and the cell bodies in the substantia nigra (E,G). Optical density analysis using ImageJ for the three proteins GFP, Ox42 and TH are presented in (H–J,L), respectively. (K) The response to 2.5 mg/kg d-amphetamine was averaged over 90 min. The dotted red line indicates the threshold of well lesioned animals. (M) Quantifications of TH-ir cells in the substantia nigra, expressed as percentage of the injected side compared to the uninjected side. Midbrain dopamine cells do not show obvious signs of degeneration (N,o) and there is only a mild inflammation response at the site of the injection as indicated by the microglial marker Ox42/cd11b (P,q).