Effective Depletion of Pre-existing Anti-AAV Antibodies Requires Broad Immune Targeting

Pre-existing antibodies (Abs) to AAV pose a critical challenge for the translation of gene therapies. No effective approach is available to overcome pre-existing Abs. Given the complexity of Ab production, overcoming pre-existing Abs will require broad immune targeting. We generated a mouse model of pre-existing AAV9 Abs to test multiple immunosuppressants, including bortezomib, rapamycin, and prednisolone, individually or in combination. We identified an effective approach combining rapamycin and prednisolone, reducing serum AAV9 Abs by 70%–80% at 4 weeks and 85%–93% at 8 weeks of treatment. The rapamycin plus prednisolone treatment resulted in significant decreases in the frequency of B cells, plasma cells, and IgG-secreting and AAV9-specific Ab-producing plasma cells in bone marrow. The rapamycin plus prednisolone treatment also significantly reduced frequencies of IgD−IgG+ class-switched/FAS+CL7+ germinal center B cells, and of activated CD4+ T cells expressing PD1 and GL7, in spleen. These data suggest that rapamycin plus prednisolone has selective inhibitory effects on both T helper type 2 support of B cell activation in spleen and on bone marrow plasma cell survival, leading to effective AAV9 Abs depletion. This promising immunomodulation approach is highly translatable, and it poses minimal risk in the context of therapeutic benefits promised by gene therapy for severe monogenetic diseases, with a single or possibly a few treatments over a lifetime

Neu5Gc expression in GRMD muscle at different ages.

<p>Three different GRMD cases (GRMD1, 2, and 3) were biopsied and muscles stained for Neu5Gc at 3, 6 and 13 months of age. Time-matched images of staining of the cranial sartorius (A) and vastus lateralis (B) muscles are shown. Bar is 50 µm for all panels in A and B.</p

Comparison of CT1 and CT2 immunostaining in DMD and normal human muscle.

<p>CT1 and CT2 were used to immunostain two different DMD (A) and two different normal human (B) muscle biopsies, compared to secondary antibody control. These were compared to time-matched images taken from mdx muscles (C), diaphragm (left panel) or tibialis anterior (right panel), that had been infected with rAAV(rh.74).MCK.GALGT2 for 12 weeks. Bar is 200 µm for all panels in A, B and C.</p

Co-localization of Neu5Gc staining with markers for endosomes and Golgi in BMD and DMD muscle.

<p>(A) BMD muscle co-stained for Neu5Gc (green) and clathrin (red), a marker of endosomes. Merged image on right shows overlap of Neu5Gc and clathrin expression in yellow. Arrow marks several examples of co-staining. (B) DMD muscle co-stained for Neu5Gc (green) with LAMP1, a lysosomal marker, 58K Golgi, a Golgi marker, or calnexin, and endoplasmic reticulum marker, all in red, and DAPI (blue). Arrow marks region of coincident staining (yellow) for Neu5Gc and 58K Golgi. Bar is 50 µm for all panels in A and B.</p

Neu5Gc co-staining with β spectrin, CD11b, CD8 or Pax7 in normal, BMD or DMD human muscle.

<p>(A) Otherwise normal, Becker muscular dystrophy (BMD) and Duchenne muscular dystrophy (DMD) muscle biopsy sections were stained for Neu5Gc (green), β spectrin (red) and DAPI (blue). Arrows indicate Neu5Gc puncta in cytoplasmic or perimembranous regions of BMD and DMD skeletal myofibers. (B) DMD muscle was co-stained for Neu5Gc and CD11b, CD8 or Pax7 (and DAPI). Bar is 50 µm for all panels in A and B.</p

Co-staining of CT carbohydrate with macrophages in GRMD muscle.

<p>(A) Single and merged staining for CT2 (green) with CD11b (red, to mark macrophages) and DAPI (blue). Arrow shows co-incident staining for CT2 and CD11b. (B) Single and merged staining for CT2 (green) with Pax7 (red, to mark satellite cells) and DAPI (blue). Arrow shows lack of coincident staining for CT2 and Pax7. (C) Examples of WFA staining (green) with embryonic myosin (red) and DAPI (blue) (two left panels). Examples of control staining for staining shown in A and B using only secondary antibodies with DAPI (two right panels). Bar is 50 µm for all panels in A, B and C.</p

Quantification of Neu5Ac and Neu5Gc levels in normal and dystrophin-deficient mouse and dog muscles.

<p>Tibialis anterior (TA) and soleus (Sol) muscles were analyzed from 11 wild type (WT) mice, TA and gastrocnemius (GS) muscles were analyzed from 8 mdx mice, and vastus lateralis (VL) and cranial sartorius (CS) muscles were analyzed from 2 golden retriever (GR) and 5 golden retriever muscular dystrophy (GRMD) dogs. Average Neu5Gc as a percentage of total sialic acid is shown. Errors are standard deviation (SD). ***P<0.001, for all dog vs. mouse muscle comparisons.</p

Quantification of CD4−, CD8−, CD11b− and Pax7−stained cells in GR and GRDM muscle and of Neu5Gc co-staining in GRMD muscle.

<p>(A) Cranial sartorius muscle sections from GR and GRMD dogs were stained with markers for satellite cells (Pax7), T lymphocytes (CD4 or CD8) or macrophages (CD11b) and quantified for numbers of cells stained per 40X visual field. (B) The percentage of cells co-stained for Neu5Gc and CD4, CD8, CD11b or Pax7 in GRMD muscles was quantified. Errors are SEM. ***P<0.001, for each GR vs. GRMD comparison in A.</p

Neu5Gc expression in Golden Retriever cross (GR) and Golden Retriever Muscular Dystrophy (GRMD) dog skeletal muscle relative to mouse.

<p>(A) Neu5Gc-specific affinity-purified chicken IgY was used to stain skeletal muscles from 6 month-old normal Golden Retriever (GR) or Golden Retriever Muscular Dystrophy (GRMD) dogs. An example of staining from a severely affected and a mildly affected GRMD dog are shown. (B) Time-matched images in A were compared to normal mouse skeletal muscle immunostained with the same reagents. Non-immune chicken IgY was used as a control for background staining in A and B. Bar is 200 µm for all panels in A and B.</p

Co-staining of CT1 with βspectrin in BMD muscles.

<p>Sections from DMD, BMD and normal human muscle biopsies sections were co-stained with CT1 (green), β spectrin(red) and DAPI (blue). Merged sarcolemmal membrane staining for CT1 and β spectrin is orange-yellow. PN represents intramuscular peripheral nerve. Bar is 50 µm for all panels.</p