Delivery of the 7-dehydrocholesterol reductase gene to the central nervous system using adeno-associated virus vector in a mouse model of Smith-Lemli-Opitz Syndrome

Abstract

Smith Lemli Opitz syndrome (SLOS) is an inherited malformation and mental retardation metabolic disorder with no cure. Mutations in the last enzyme of the cholesterol biosynthetic pathway, 7-dehydrocholesterol reductase (DHCR7), lead to cholesterol insufficiency and accumulation of its dehyrdocholesterol precursors, and contribute to its pathogenesis. The central nervous system (CNS) constitutes a major pathophysiological component of this disorder and remains unamenable to dietary cholesterol therapy due to the impenetrability of the blood brain barrier (BBB). The goal of this study was to restore sterol homeostasis in the CNS. To bypass the BBB, gene therapy using an adeno-associated virus (AAV-8) vector carrying a functional copy of the DHCR7 gene was administered by intrathecal (IT) injection directly into the cerebrospinal fluid of newborn mice. Two months post-treatment, vector DNA and DHCR7 expression was observed in the brain and a corresponding improvement of sterol levels seen in the brain and spinal cord. Interestingly, sterol levels in the peripheral nervous system also showed a similar improvement. This study shows that IT gene therapy can have a positive biochemical effect on sterol homeostasis in the central and peripheral nervous systems in a SLOS animal model. A single dose delivered three days after birth had a sustained effect into adulthood, eight weeks post-treatment. These observations pave the way for further studies to understand the effect of biochemical improvement of sterol levels on neuronal function, to provide a greater understanding of neuronal cholesterol homeostasis, and to develop potential therapies.

Keywords: 7-dehydrocholesterol reductase (DHCR7); AAV; Smith Lemli Opitz syndrome (SLOS); central nervous system (CNS); gene therapy; intrathecal.

Fig. 1

Kinetics of 7-DHC/cholesterol in the brain (A), spinal cord (B) and sciatic nerve (C) of SLOS mice. For each time point, the ratio is an average of 6 animals and the bars represent the standard error of the mean (SEM). A higher 7-DHC/cholesterol ratio is indicative of a greater biochemical imbalance. This ratio is very low (close to zero) in normal mice and does not change with age.

Fig. 2

IT injection into the CSF in a 3 day old mouse pup. Point of injection is indicated and delivery of dye to the spinal cord and brain is observed.

Fig. 3

Detection of human DHCR7 DNA in brain 8-week after IT delivery of AAV vectors. (A) The hDHCR7 specific band (123 bp) is seen only in IT injected animals (lanes 1–5), and is absent in the saline injected counterparts (lanes 6–10). Lane 11 represents a non-template control. (B) All brain DNA preparations show the presence of the reference nuclear gene Fabpi (the order of lanes is the same as in A except that lanes 10 and 11 are reversed).

Fig. 4

Expression of mRNA in brain. RNA extracts from brains of AAV-DHCR7 treated (lanes 1–11) and saline treated (lanes 12–17) mice were tested for hDHCR7 mRNA (A) and mouse GAPDH mRNA (B). Non-reverse transcriptase treated samples showed the absence of hDHCR7 or GAPDH specific PCR product confirming that the amplification products result from mRNA derived cDNA rather than from the presence of contaminating DNA (data not shown).

Fig. 5

Partial normalization of cholesterol metabolism. DHC/cholesterol in the brain (A) and spinal cord (B) of treated (n = 11) and untreated (n = 5) SLOS mice and their normal (T93M/+) littermates (n = 6). SLOS mice were treated IT with 30 μl of AAV-DHCR7 or saline at 3 days of age and sterol analysis by GC/MS was performed at 8 weeks post-treatment. DHC is the sum of 7-DHC and 8-DHC. Statistical analyses were performed using the non-parametric Mann–Whitney test.

Fig. 6

Correlation of DHC/C ratios with hDHCR7 DNA and mRNA in brain. (A) Transgene (hDHCR7) copy number versus DHC/C shows an exponential relationship with a correlation coefficient of r² = 0.82. (B) Relative hDHCR7 mRNA levels expressed as 2^{− ΔCt (Ct}_(hDHCR7)^− Ct_(mGAPDH)⁾ versus DHC/cholesterol similarly shows an exponential relationship with a correlation coefficient of r² = 0.73.

Fig. 7

Partial normalization of cholesterol metabolism in sciatic nerve. Ratios of DHC/cholesterol in the sciatic nerve of SLOS mice treated IT with AAV-DHCR7 (n = 11), or saline (n = 5) and their normal littermates (n = 6) were compared. Tissues were collected 8 weeks after treatment of 3-day old mice, DHC is the sum of 7-DHC and 8-DHC, and the non-parametric Mann–Whitney test was used for statistical analysis.

Fig. 8

Sterol analysis in the liver of IT-treated SLOS mice. The 7-DHC/cholesterol ratio remains unchanged in AAV-DHCR7 (n = 9) versus saline (n = 5) at 8 weeks post-treatment. As 8-DHC levels are negligible in the liver, only 7-DHC levels were measured. Statistical analysis was performed using the non-parametric Mann–Whitney test.