AAV-serotype-specific Transduction Patterns In Mice And Non-human Primates (NHPs) Liver Tissue: Implications For Therapeutic Efficacy

Anna Majowicz, Lukas K Schwarz, Johannes PF de Laat, Sander J van Deventer and Valerie Ferreira

Introduction / Background

AAV-based liver gene therapy has proven efficacious in mouse models of inherited disorders, but little is known about the transduction pattern of various

AAV serotypes in the primate or human liver. To address this question, we assessed the AAV distribution pattern in the liver tissue of mice and non-human

primates (NHPs) injected with either AAV serotype 1 or 5ch

(chimeric AAV5 in which VP1-unique portion is of AAV2 origin). The overall percentage of cells

positive for the presence of AAV vector DNA/hFIX transgene RNA as well as the intensity and area of the positive signal were assessed. Additionally, AAV

vector spatial distribution throughout the liver tissue was determined.

Results In mice (I) the total percentage of liver cells positive for the presence of AAV vector DNA/hFIX mRNA was lower for AAV1 (Mean= 32.44 %; n=3) than for

AAV5ch

(Mean= 56.08 %; n=4) (Ia). Also, liver cells positive for AAV5ch

vector DNA/hFIX RNA displayed a higher signal score, thus higher H-score, than

liver cells positive for AAV1 vector DNA/hFIX RNA suggesting more efficient transduction per cell by the AAV5ch

vector (I b). The majority of cells

transduced by AAV1 or AAV5ch

(>99%) expressed Albumin and therefore were characterized as hepatocyte. Interestingly, the spatial distribution of AAV

vector DNA/hFIX RNA positive signal within the liver tissue was different for the two serotypes: AAV5ch

vector DNA/hFIX RNA (visualized in red) was

more localized around the central veins (visualized in yellow by GS IHC) (I c,e) whereas AAV1 was more homogenously distributed throughout the liver

tissue (I d,e).

In NHPs (II), the percentage of liver cells positive for AAV vector DNA/hFIX RNA was also higher for AAV5ch

(Mean= 45.53%; n=2) than for AAV1 (Mean=

26.3 %; n=3) (II a), and similar to mice, AAV5ch

resulted in a higher AAV vector DNA/hFIX RNA probe signal score, thus H-score, indicating a more

efficient transduction per cell with AAV5ch

than with AAV1 in injected NHPs (II b). Detailed analysis of AAV vector spatial distribution throughout the NHP

liver tissue demonstrates differences between mice and NHPs in AAV distribution pattern for AAV5ch

vector. In NHPs, not only AAV1 but also AAV5ch

vector DNA/transgene RNA (visualized in red) was homogenously distributed throughout the liver tissue (II c,d,e).

AAV1-hFIX: DAPI, hFIX FISH & GS IHC

Study design C57BL/6 mice were injected intravenously (IV) with either AAV1-hFIX (human factor IX), AAV5

ch-hFIX at dose 1.46 e13 gc/kg or PBS, while NHPs were

injected with either AAV1-hFIX, AAV5ch

-hFIX at dose 3e13 gc/kg or PBS. Liver tissues were collected post mortem and OCT frozen liver pieces for mice

while FFPE liver pieces for NHPs were analyzed by fluorescent in situ hybridization (FISH) using fluorescent probes specific for AAV vector DNA and hFIX

transgene mRNA. Hepatocytes were characterized based on Albumin RNA expression in mice and Serpina1 RNA expression in NHPs (FISH) while

central veins were localized based on Glutamine Synthetase (GS) protein as determined by immunohistochemistry (IHC). FISH to detect Albumin RNA in

mice or Serpina1 in NHPs, FISH for hFIX AAV vector DNA/transgene RNA and IHC for GS were performed on the same sections. Images were acquired

with Aperio Versa 8 slide scanner (Leica Biosystems) and analyzed with the use of an image analysis software (HALO, indica labs). For FISH image

analysis, cells were scored from weak positive (1+) to strong positive (4+) based on combination of average positive signal area [µm2] and average

intensity of positive signal within cell [RFU]. Based on the percentage of scored cells the H-score is calculated (H-score= % of “1+” cells + 2* % of “2+”

cells + 3* % of “3+” cells + 4* % of “4+” positive cells) that can range between 0 (if all cells are negative) and 400 (if all cells are “4+” strong positive).

Conclusions

In summary, we observed remarkable differences in AAV5ch

transduction profiles in liver tissue of mice

and NHPs. In mice AAV5ch

vector DNA/hFIX transgene RNA was more localized around central veins in the

liver while in NHPs it was homogenously distributed throughout liver tissue. These results indicate that

mouse models may have a limited value to predict the efficacy of liver-targeted AAV-based gene therapy,

in particular in the context of development of therapies for metabolic disorders.

b a

II

b a

d c d c

e e

I

Research and Development

Amsterdam, The Netherlands

+31 202406023

a.majowicz@uniqure.com

II

AAV1-hFIX: DAPI & hFIX FISH

AAV1-hFIX: DAPI, hFIX FISH & GS IHC AAV5ch-hFIX: DAPI, hFIX FISH & GS IHC

AAV5ch- hFIX: DAPI & hFIX FISH

AAV5ch-hFIX: DAPI, hFIX FISH & GS IHC

AAV1-hFIX: DAPI & hFIX FISH AAV5ch- hFIX: DAPI & hFIX FISH

AAV1: n=3 mice, 119 CV +

173 PV analyzed

AAV5ch

: n=4 mice, 328 CV

+ 566 PV analyzed

AAV1: n=3 NHP, 221 CV +

229 PV analyzed

AAV5ch

: n=2 NHP, 86 CV +

133 PV analyzed