Imaging gene delivery in a mouse model of congenital neuronal ceroid lipofuscinosis

Abstract

Adeno-associated virus (AAV)-mediated gene replacement for lysosomal disorders have been spurred by the ability of some serotypes to efficiently transduce neurons in the brain and by the ability of lysosomal enzymes to cross-correct among cells. Here, we explored enzyme replacement therapy in a knock-out mouse model of congenital neuronal ceroid lipofuscinosis (NCL), the most severe of the NCLs in humans. The missing protease in this disorder, cathepsin D (CathD) has high levels in the central nervous system. This enzyme has the potential advantage for assessing experimental therapy in that it can be imaged using a near-infrared fluorescence (NIRF) probe activated by CathD. Injections of an AAV2/rh8 vector-encoding mouse CathD (mCathD) into both cerebral ventricles and peritoneum of newborn knock-out mice resulted in a significant increase in lifespan. Successful delivery of active CathD by the AAV2/rh8-mCathD vector was verified by NIRF imaging of mouse embryonic fibroblasts from knock-out mice in culture, as well as by ex vivo NIRF imaging of the brain and liver after gene transfer. These studies support the potential effectiveness and imaging evaluation of enzyme replacement therapy to the brain and other organs in CathD null mice via AAV-mediated gene delivery in neonatal animals.

Figure 1. AAV constructs and gene delivery of CathD to knock-out MEFs

(a) Maps of pAAV constructs expressing mouse CathD (mCathD) cDNA or GFP under control of the strong constitutively active CBA promoter. (b) MEF −/− knock-out cells were transfected with pAAV-mCathD or pAAV-GFP. Forty-eight h later, cells were lysed and analyzed by SDS-PAGE and western blotting using anti-CathD antibody and β-actin for loading efficiency. Showing is a representative blot from 3 independent experiments all yielding similar results.

Figure 2. NIRF imaging of CathD in cultured cells

(a) Knock-out (−/−) MEFs were transfected with pAAV-GFP and 48 hours later analyzed for GFP expression. Typical dot-plot showing the transfection efficiency of MEFs being around 18%. (b–c) knockout cells transfected with pAAV-GFP or both pAAV-mCathD and pAAV-GFP. Forty-eight h later, these cells as well as plain knockout and wild-type (+/+) MEFs were incubated with the CathD-specific NIRF probe and analyzed by FACS 1 h later.(b) Representative histograms showing an intense Cy5.5 fluorescence in knockout MEFs transfected with pAAV-mCathD and pAAV-GFP as well as in wild-type MEFs, but not in knock-out MEFs transfected with pAAV-GFP. (c) Quantitation analysis of FACS data from three independent experiments. MFI = mean fluorescence intensity.

Figure 3

AAV2/rh8-mCathD vector provides efficient gene delivery and increased survival of knock-out mice.(A) Neonatal pups were injected with PBS or AAV2/rh8-mCathD vector both i.c.v. (2 × 10¹⁰ g.c to each ventricle in 2 μl 0.06% Trypan blue) and i.p. (1 × 10¹⁰ g.c in 100 μl) once between P1-4 (n = 10). Time of death was recorded. The data is presented as a Kaplan-Meier survival curve of CathD knock-out mice from both groups using JMP Software. Log-rank, as well as Wilcoxon test was performed to compare mean survival of AAV2/rh8-mCathD-treated mice versus PBS control knock-out mice and the extension of survival by treatment with AAV2/rh8-mCathD was significant (Log-Rank p≤0.02; Wilcoxon p≤0.05.). The same experiment was repeated three times and similar results were obtained. (B) Neonatal pups were injected with AAV2/rh8-mCathD at P1 as in (A) and sacrificed at P27. Brain from these mice as well as from non-injected knockout, heterozyous and wild-type mice were removed, lysed and 50 μg protein was analyzed by western blotting for mCathD expression. The short live 53 kDa precursor of CathD is processed into the 47 kDa intermediate form and the mature enzyme composed of 31 kDa and 14 kDa fragment.

Figure 4

NIRF imaging of CathD activity ex vivo. Neonatal pups were injected with AAV2/rh8-mCathD vector both i.c.v. (2 × 10¹⁰ g.c into each ventricle in 2 μl 0.06% Trypan blue) and i.p. (1 × 10¹⁰ g.c in 100 μl) on P1. Similar groups of mice were injected with PBS (n = 6/group). On P24 (2–3 days prior to mortality in knock-out animals), mice were injected i.p. with NIRF probe (0.1 nmoles/g body weight) and sacrificed 24 h later with PBS perfusion. Liver, brain, heart, spleen and kidneys were removed, rinsed with PBS, and imaged at 5 different regions of interests using a custom-built camera system followed by image analysis using Kodak Digital Science 1D software. B).(A) representative organs from a single mouse in each group is shown.(B) Signal intensity is expressed as the average relative light units (RLU) ± S.D. (n = 6). Significant increases in AAV-mCathD treated animals were seen for liver (*p<0.0001) and brain (**p<0.00001) as calculated by student t-test.