The AAV2.7m8 capsid packages a higher degree of heterogeneous vector genomes than AAV2

Abstract

Recombinant adeno-associated virus (rAAV) vectors are currently the only proven vehicles for treating ophthalmological diseases through gene therapy. A wide range of gene therapy programs that target ocular diseases are currently being pursued. Nearly 20 years of research have gone into enhancing the efficacy of targeting retinal tissues and improving transgene delivery to specific cell types. The engineered AAV capsid, AAV2.7m8 is currently among the best capsids for transducing the retina following intravitreal (IVT) injection. However, adverse effects, including intraocular inflammation, have been reported following retinal administration of AAV2.7m8 vectors in clinical trials. Furthermore, we have consistently observed that AAV2.7m8 exhibits low packaging titers irrespective of the vector construct design. In this report, we found that AAV2.7m8 packages vector genomes with a higher degree of heterogeneity than AAV2. We also found that genome-loaded AAV2.7m8 stimulated the infiltration of microglia in mouse retinas following IVT administration, while the response to genome-loaded AAV2 and empty AAV2.7m8 capsids produced much milder responses. This finding suggests that IVT administration of AAV2.7m8 vectors may stimulate retinal immune responses in part because of its penchant to package and deliver non-unit length genomes.

Fig. 1. Production and analyses of AAV2 and AAV2.7m8 test vectors.

a Diagrams of self-complementary (sc)AAV-CB6-Egfp, single-strand (ss) AAV-CB6-Egfp, and ssAAV-CB6-FLuc vectors. b Photographs of the three vectors subjected to CsCl density gradient ultracentrifugation. Bands representing empty capsids (light-blue arrowheads) and full capsids (purple arrowheads) are indicated. c ddPCR quantification of scAAV-Egfp and ssAAV-Egfp vectors using probe sets against the CB6 promoter and the Egfp transgene displayed as a stacked histogram. Detection of CB6-only (red) or Egfp-only (green) droplets indicate partial genomes, while detection of both CB6 and Egfp (blue) species suggests the presence of full-length genomes. Values represent mean frequencies ± SD, (n = 3). The mean percentages of droplets double positive for CB6 and Egfp probe signals are shown. Diagram of the vector genome target with the approximate position of the probes is shown above. d Alkaline gels for AAV2 and AAV2.7m8 vectors packaged with scAAV-CB6-Egfp (left), ssAAV-CB6-Egfp (center), and ssAAV-CB6-FLuc (right). Bands predicted to contain unit length genomes are marked with purple arrowheads.

Fig. 2. 7-mer inserts with AAV2 drive the packaging of non-unit length vector genomes, but not with AAV9.

DNase-resistant genomes were isolated from vectors and ran on alkaline gels. Gels for scCB6-Egfp vectors packaged with AAV2 and three additional retinotropic 7-mer peptide insert capsids (a), for ssCB6-mCherry vectors packaged with AAV9 or MyoAAV capsids (b), and for ssEgfp vectors packaged with AAV9 or AAV9.PHP.eB capsids (c) are displayed. Each gel was accompanied by a 1-kb DNA ladder. The expected full-length genome bands are marked with magenta arrowheads.

Fig. 3. AAV-GPseq analyses of vector genomes packaged with AAV2 and AAV2.7m8 capsids.

a, c, e IGV displays of SMRT sequencing reads representing scAAV-CB6-Egfp (a), ssAAV-CB6-Egfp (c), and ssAAV-CB6-FLuc (e) vector genomes packaged into AAV2 (left) or AAV2.7m8 (right) aligned to their respective cis-plasmid references. Alignments are shown in squished displays with soft-clipped bases shown. Regions of read matches (gray), mismatches (colored), and insertions/deletions (speckles) are shown. Alignment coverages are shown above each display. b, d, f Traces of relative read abundances as a function of read length for scAAV-CB6-Egfp (b), ssAAV-CB6-Egfp (d), and ssAAV-CB6-FLuc (f) vector genomes packaged with AAV2 (blue traces) or AAV2.7m8 (red traces). The read lengths are normalized and scaled to the highest peak set to 100. Counts are binned into 10-nt distributions. Peaks representing the expected full-length genomes are marked with a purple arrowhead.

Fig. 4. Quantification of microglia in capsid/vector-treated retinas.

a, b Immunofluorescence imaging of representative retinal cross-sections from mouse eyes IVT injected with empty AAV2 or AAV2.7m8 capsids (a), and eyes injected with scAAV2-Egfp or scAAV2.7m8-Egfp vectors (b). Sections were stained to visualize EGFP (anti-EGFP, green), DNA (DAPI, blue), cone photoreceptors (PNA, white), and microglia (IBA1, red). Scale bar = 100 μM. c Quantification of IBA+ cells in capsid/vector-treated retinas. Saline group, n = 3; empty capsid groups, n = 5; Egfp vector groups, n = 7. *p < 0.05, one-way ANOVA with Tukey’s method for multiple comparisons. d Counts of microglia (IBA1+) in each retinal layer were tabulated for three cross-sections from different eyes of each treatment group. Photoreceptor segment layer (PS); outer nuclear layer (ONL); outer plexiform layer (OPL); inner nuclear layer (INL); inner plexiform layer (IPL); ganglion cell layer (GCL); shapes (circles, squares, and Xs) represent counts from each cross-section. The plot is accompanied by an example cross-section of a vector-treated retina (scAAV2-Egfp) to indicate layers of the retina.