AAV9-NPC1 significantly ameliorates Purkinje cell death and behavioral abnormalities in mouse NPC disease[S]

Niemann-Pick type C (NPC) disease is a fatal inherited neurodegenerative disorder caused by loss-of-function mutations in the NPC1 or NPC2 gene. There is no effective way to treat NPC disease. In this study, we used adeno-associated virus (AAV) serotype 9 (AAV9) to deliver a functional NPC1 gene systemically into NPC1−/− mice at postnatal day 4. One single AAV9-NPC1 injection resulted in robust NPC1 expression in various tissues, including brain, heart, and lung. Strikingly, AAV9-mediated NPC1 delivery significantly promoted Purkinje cell survival, restored locomotor activity and coordination, and increased the lifespan of NPC1−/− mice. Our work suggests that AAV-based gene therapy is a promising means to treat NPC disease.

Niemann-Pick type C (NPC) disease is an autosomal recessive lysosomal storage disorder that primarily strikes children with featured symptoms such as cerebellar ataxia, dementia, dysphagia, dysarthria, hepatosplenomegaly, and premature death early in life. It results from loss-of-function mutations in NPC1 (95% of cases) or NPC2 (5% of cases), which encode a large polytopic membrane protein and a small luminal protein, respectively (1)(2)(3). In the lysosome, NPC2 binds and delivers LDL-derived cholesterol to the N-terminal domain of NPC1 (4). The NPC1-bound cholesterol then penetrates through the glycocalyx and 4% paraformaldehyde (PFA) and subjected to following staining processes (22).

Immunocytochemistry
Twenty-four hours after plasmid transfection, cells were fixed with 4% PFA in PBS for 30 min at room temperature and washed twice with PBS. Cells were incubated with 50 g/ml filipin and 10 g/ml anti-Flag antibody in the dark for 1 h, washed with PBS three times, and incubated with Alexa Fluor 488 goat anti-mouse IgG for 1 h at room temperature. Cells were examined and imaged under a Leica TCS SP5 confocal microscope.

Histochemistry
Mouse tissues were fixed in 4% PFA, embedded in paraffin, and cut into 3 m sections using a microtome (Leica RM2235) or embedded in OCT for frozen section with a cryostat (Leica CM 3050S). For histological analysis, paraffin sections were deparaffinized and stained with H&E (Sigma). For immunostaining analysis, the sections were processed as previously described (23). Briefly, deparaffinized sections were boiled (95°C) for 15 min in 25 mM Tris-HCl and 1 mM EDTA buffer (pH 9.0) for antigen retrieval. The same procedures followed afterwards for both paraffin sections and frozen sections. Samples were then permeabilized and blocked with 5% FBS in PBS containing 0.5% Triton X-100 for 1 h at room temperature. Sections were incubated with primary antibodies overnight at 4°C and then washed with PBS three times followed by incubation with the appropriate secondary antibodies for 1 h at room temperature. Sections were finally incubated with 5 g/ml Hoechst for 5 min and mounted.

Western blotting
Mouse tissues were snap-frozen and homogenized in 1 ml of RIPA buffer containing protease and phosphatase inhibitors with a high-throughput tissue homogenizer (Bertin Technologies; Pre-cellys®24) as previously described (23,24). Lysate was centrifuged at 10,000 g for 10 min at 4°C and protein concentrations were determined using the Lowry method (Bio-Rad). Samples were mixed with the loading buffer and boiled for 10 min. Proteins were resolved by SDS-PAGE electrophoresis and transferred onto nitrocellulose membranes. Membranes were blocked with 5% BSA in TBS containing 0.075% Tween-20 (TBST) and probed with primary antibodies overnight at 4°C. After washing in TBST three times, blots were incubated with secondary antibodies for 1 h at room temperature followed by three more washes in TBST. Immunoblots were analyzed with a Tanon 5200 chemiluminescent imaging system (Shanghai China).
P4 mice were anesthetized on wet ice. A total volume of 25 l containing 2.5 × 10 11 vg AAV9-EGFP or AAV9-NPC1 viruses was intracardially injected into the left ventricle using a 29 gauge insulin syringe (Ultral fine needle; BD). Mouse tissues were collected for Western blotting and histochemical analysis 8 weeks postinjection.

Open field test
A 40 cm × 40 cm field was equally divided into 3 × 3 squares with the middle square designated as the center. Mice dotted with crystal violet staining solution on their back were put onto the middle (3.9 kb) open reading frame poses a challenge to deliver a functional NPC1 gene by AAV in vivo.
Here, we successfully packaged the expression cassette of NPC1 into a standard AAV9 vector. A single systemic injection of AAV9-NPC1 into NPC1 / mice at postnatal day 4 (P4) resulted in high expression of NPC1 protein in the brain, lung, heart, and other peripheral tissues. Moreover, Purkinje cell survival, behavioral abnormalities, and lifespan were greatly improved in NPC1 / mice following AAV9-NPC1 treatment. Together, our work establishes AAV9-mediated NPC1 delivery as a novel and promising approach to treat NPC disease.

Animals
All animal experiments were approved by the Biological Research Ethics Committee, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. NPC1 / and WT littermates were bred using heterozygous pairs (BALB/cNctr-Npc1m1N/J) purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were fed on a chow diet ad libitum and housed in a pathogenfree animal facility in plastic cages at 22°C, with a daylight cycle from 6:00 AM to 6:00 PM. Pups were genotyped at 2 days of age. At 63 days of age, mice were subjected to the open field test and rotarod test.
We then incorporated NPC1-3×Flag into a standard AAV9 vector and intracardially injected AAV9-NPC1 into the heart of NPC1 / mice at P4. Each mouse received a single injection of 25 l containing 2.5 × 10 11 vg of virus, and the expression levels of NPC1 in different tissues were analyzed 2 months postinjection. The validity and specificity of M2 anti-Flag antibody was confirmed (supplemental Fig. of a piece of white paper covering the entire field and allowed to explore freely for 10 min. The movement was recorded by an overhead camera and the total distance traveled and time spent in the center were analyzed using EthoVision XT 10 (Noldus, Leesburg, VA).

Rotarod test
The balance and motor coordination of mice were tested on a rotarod machine (ZB_200; Chengdu Techman Software Co., Ltd.) as previously described (25).

Statistics
Results are presented as mean ± SD. All the data were analyzed by unpaired two-tailed Student's t-test or one-way ANOVA. Statistical significance was set at P < 0.05.

Generation of AAV9-based NPC1 construct
To engineer a NPC1 expression cassette that would fit the packaging capacity (4.5-5.0 kb) of AAV9 vector, we employed a small-sized (664 bp) CMV promoter with high transcription activity NPC1 coding sequence fused with 3×Flag instead of fluorescent protein at the C terminus, and a 48 bp miniature poly(A) signal, rather than the commonly used hGH poly(A) signal (500 bp). The final CMV-NPC1-3×Flag-miniPolyA vector was 4.74 kb in size and appropriate for AAV9 packaging (Fig. 1A).
We next sought to test whether the NPC1-3×Flag construct would express a functional NPC1 protein. CT43 cells are deficient in NPC1 and display excess cholesterol in lysosomes (26). Overexpression of NPC1-3×Flag, however,  Calnexin is used as a loading control. B: Confocal images of exogenous NPC1 staining (red) in the brain, heart, and lung. Nuclei were counterstained with Hoechst (blue). Boxed areas in the left column are shown at a higher magnification in the right column. lysosome patterning in the cells of the abovementioned tissues.

AAV9-NPC1 injection dramatically improves the survival of Purkinje cells
Purkinje cell loss, which initiates from 49 days of age and becomes pronounced by 9 weeks of age in NPC1 / mice, is a hallmark of NPC disease (10,27,28). To evaluate the effect of AAV9-NPC1 injection on Purkinje cell survival, we administered AAV9-EGFP or AAV9-NPC1 systemically into NPC1 / mice at P4 and examined cerebellar Purkinje cells 2 months postinjection. A severe loss of Purkinje cells was observed in NPC1 / mice injected with AAV9-EGFP ( Fig. 3A-D). AAV9-NPC1 treatment, however, significantly S2). High levels of mature glycosylated NPC1 expression were evident in the lung, brain, heart, and spleen, followed by less robust, but still apparent, signal in the liver, kidney, and colon ( Fig. 2A). A minimal amount of NPC1 was present in the stomach, jejunum, and muscle ( Fig. 2A). Immunohistochemical analysis revealed that AAV9-delivered NPC1 primarily localized in the outer cerebral cortex, whereas the distribution of NPC1 expression was relatively homogeneous throughout the heart and lung (Fig. 2B). A similar pattern was also observed following AAV9-EGFP injection (supplemental Fig. S1A, B), suggesting that the delivery method, but not exogenous protein per se, determines distributions of the proteins. Importantly, ectopic NPC1 appeared to form cytoplasmic puncta and correspond to in neurons and astrocytes effectively corrects the neurological signs and prolongs the lifespan of NPC1 / mice, suggesting an interesting possibility of re-expressing a functional NPC1 protein in the CNS by gene therapy to treat NPC disease.
An ideal gene therapy would correct the disease-causing mutations in every single cell. Although new powerful gene editing tools, such as TALEN and CRISPR/Cas9, are emerging, precise DNA repair is still difficult to achieve and only feasible in the zygotes and liver where highly efficient homologous recombination occurs (31)(32)(33)(34). Because the direct DNA repair of mutant NPC1 in the CNS is hardly approachable, we thus took the traditional gene therapy strategy to treat recessive inherited NPC disease by re-expressing functional copies of the target gene (16,17).
Nevertheless, how to assure specific delivery and the stable long-term expression of transgenes remains challenging. Several vectors, such as adenovirus, lentivirus, and AAV, have been used for in vivo gene delivery. Lentivirus can integrate into the genomic DNA of target cells, but improved Purkinje cell survival, as revealed by calbindin labeling (Fig. 3E, F) and H&E staining (Fig. 3G, H). Quantification showed that the number of Purkinje cells increased by about 160% in AAV9-NPC1-injected mice compared with that of AAV9-EGFP-injected mice (Fig. 3I).

AAV9-NPC1 injection rescued the gene expression abnormalities in liver and brain of NPC1 / mice
Deficiency in NPC1 or NPC2 impedes the lysosome-to-ER transport of LDL-cholesterol; therefore, it activates the expression of the genes involved in cholesterol biosynthesis (7). In addition, a compensatory increase in lysosomal genes was observed due to lysosomal dysfunctions (9,29). Inflammation was upregulated in NPC1-deficient mice. We then examined gene expression profiles of the abovementioned biological events. RT-quantitative (q)PCR revealed a significant decrease in the genes involved in cholesterol metabolism (ApoB, HMGCS), lysosome (LIMP1, cathepsin L1, cathepsin B, acid lipase), and inflammation (TNF, CCL5) in the liver of NPC1 / mice receiving AAV9-NPC1 injection compared with those receiving AAV9-EGFP injection (Fig. 4A). Similar effects were also observed in brain tissue (Fig. 4B).

AAV9-NPC1 injection significantly ameliorated the motor deficits of NPC1
/ mice We next monitored the general activity and motor coordination of WT littermates and NPC1 / mice injected with AAV9-EGFP or AAV9-NPC1 using the open field paradigm. NPC1 / mice were less active and showed defects in moving, standing, and taking food at the age of 9 weeks (data not shown). Compared with WT controls that primarily moved marginally, NPC1 / mice injected with AAV9-EGFP displayed a shorter moving distance with significantly longer periods in the center (Fig. 5A-C). These moving deficits, however, were markedly rescued by AAV9-NPC1 administration ( Fig. 5A-C). When subjected to the rotarod test, AAV9-NPC1-treated mice stayed on top of a rotating cylinder for a much longer time than those receiving AAV9-EGFP (Fig. 5D). These results indicate that AAV9-NPC1 treatment effectively ameliorates the locomotor activity and motor coordination in NPC1 / mice.

AAV9-NPC1 injection extended the lifespan of NPC1
We finally assessed whether systemic AAV9-NPC1 injection could increase the survival time of NPC1 / mice.

DISCUSSION
NPC disease is a neurodegenerative disorder with much more severe defects in the CNS than in the peripheral tissues (14,30). Replacement expression of WT NPC1 transgene Fig. 4. Intracardiac injection of AAV9-NPC1 reverses expression of associated genes. A: Quantitative PCR analysis of genes in liver of WT mice and NPC1 / mice injected with AAV9-EGFP or AAV9-NPC1. B: Quantitative PCR analysis of genes in brain of WT mice and NPC1 / mice injected with AAV9-EGFP or AAV9-NPC1. Data are presented as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, unpaired two-tailed Student's t-test.
induces insertional mutagenesis. Adenovirus, though nonintegrative and safer, produces only transient expression (35). AAV, via different serotypes, transduces nondividing cells of various tissues and results in highly efficient gene expression without genome integration (16). AAV-mediated delivery of factor IX and LDLR, which were developed for therapeutic purposes for hemophilia B and homozygous familial hypercholesterolemia, respectively, is currently in phase 1 and phase 2 clinical trials (36)(37)(38). AAV9, owing to the capability of infecting both neurons and glial cells upon intravenous injection (21), is a good candidate for transferring the NPC1 gene into the CNS of NPC1 / mice.
In this study, the lifespan of NPC1 / mice was increased by about 30% after a single intraventricular injection of AAV9-NPC1 at P4. This limited improvement is probably attributable to the restricted NPC1 expression in the outer cortex and hippocampus (supplemental Fig. S1B). We have also tried intracranial administration and found a widespread expression of NPC1 in both the cerebrum and cerebellum (supplemental Fig. S3A, B). This would be more helpful to improve longevity. In addition, a very recent study reports that retro-orbital delivery of AAV9-based NPC1 expression under CamKII or EF1a promoters substantially increases the lifespan of NPC1 / mice (39). It will be interesting to compare the effects of various delivering routes, such as intracisternal (40), intracerebroventricular (41), or intracranial injections, on NPC1 expression in the CNS, as well as the reparative effects of AAV9-NPC1 on NPC1 phenotypes. In addition, whether a combination of different types of AAVs efficiently promotes global gene delivery needs further investigation.
In summary, our study demonstrates that AAV9-delivered expression of NPC1 is an effective way to treat NPC disease.