Wide Transgene Expression In The Central Nervous System Mediated By Intrathecally Delivered RAAV9 In Mice

Objectives:The idea of treating human diseases by delivering therapeutic transgenes was first accepted in the 1970’. Then people find the possibility to use the viruses as vectors. The ideal vector must have three characters: First, high affinity and transduction efficiency to the target tissues and low transduction efficiency to the non-target cells, tissues and organs; Second, the vector could express at a sustained therapeutic level over a long period of time; Third, also the most important, minimal side effect and immunogenicity.Adeno-associated virus(AAV) is a member of the family Parvoviridae in the genus Dependovirus. Dependoviruses are non-autonomous, requiring co-infection with a helper virus in order to replicate, and non-pathogenic. These aspects of its natural biology greatly reduce the risks of side effects associated with the use of AAV for gene delivery. Recombinant AAV(r AAV) was considered as one of the most promising vectors for gene transfer, because of its safety, broad host range, minimal immunogenicity, and sustained expression of exogenous genes in vivo. And they are now widely used in the basic and clinical studies. r AAV can mediate transgene expression in the central nervous system(CNS). As a number of the r AAVs, r AAV9 can cross the brain blood barrier, and mediate transgene expression in the brain and spinal cord for a long period of time. Therefore, r AAV9 has potential advantages in the gene-based therapies for CNS disorders.The most important thing in gene therapy is to deliver transgene to enough cells affected. The distribution of transgene expression differs from different delivery methods, and different ways need different doses of the vector. Parenchymal injection can transduce the limited local areas, but not applicable to amyotrophic lateral sclerosis(ALS) in which broad areas including the whole spinal cord, brainstem and cortex are afflicted. Intravenous injection of r AAV can transduce wide areas of CNS in neonatal mice, but transduction efficiency is relatively low in adult mice, together with less targeting, requirement of high doses of vectors, and potential safety hazards. Intra-cerebrospinal fluid(CSF) injected r AAV contact CNS directly without blockade of blood brain barrier. Intra-CSF delivery methods include intracerebroventricular, intra-cisterna magna, and intrathecal injections, of which the later has more advantages on spinal cord transduction. Intrathecal injection of r AAV9 into pigs successfully transduced motor neurons in the spinal cord. Direct intrathecal injection has been widely used in the clinical pharmaceutical studies, and it is an invasive method for gene therapy. Some researchers used intrathecal catheter or direct lumbar puncture to study AAV-mediated transgene expression, a common problem existed is obvious variations in transduction extent and areas among different individuals. The authors thought that it was related to the uncertainty on the successfulness of placing catheter or intrathecal injection. Direct intrathecal injection is safer, noninvasive and simple compared with intrathecal catheter placement, however, faces the inherent technical difficulty to inject intrathecally in a small animal constant amount of r AAV vectors. The different extent of successfulness in direct intrathecal injection will lead to obvious variations in transduction efficiency and clinical effect. Therefore, a noninvasive, simple and controllable wide CNS transduction method is needed.In this study, aimed at the blindness of lumbar puncture in mice, we established a simple, efficient instant evaluating system to evaluate the effect of intrathecal injection. Using the modified direct intrathecal injection method, r AAV9-CB6-EGFP was delivered into cerebrospinal fluid(CSF) in mice, and CNS transduction areas, efficiency and cellular tropism were studied.Methods:1 Intrathecal injection in miceIntrathecal injection of PBS or r AAV9-CB6-EGFP vector in awake male FVB mice was carried out at 8-10 weeks of age. A total of 4x1010 GC vectors were injected in an 8ul volume, containing 1% lidocaine. Transient limb weakness and paralysis were recorded right after the injection. All injected mice were maintained for 3 weeks and sacrificed for fresh tissue collection or perfusion with 4% paraformaldehyde.2 ImmunohistochemistrySpinal cord and brain tissues fixed by 4% paraformaldehyde were cut into 20μm free-floating sections using a vibratome(Leica VT 1000S). The sections were rinsed for 3 times in 0.01 M PBS, then treated with 3% hydrogen peroxidase in methanol, blocking endogenous peroxidase. The sections were then perforated with 0.3% Triton X-100. After blocking with 10% horse serum at room temperature, the sections were incubated overnight at 4°C with primary antibodies including anti-GFP, anti-GFAP and anti-Iba1 antibodies. After rinsing, the sections were subsequently incubated with a biotin-conjugated secondary antibody followed by horseradish peroxidase conjugated with streptavidin at room temperature. Then the sections were treated with 0.03% diamino-benzidine as a chromogen for 50 s, spread onto the slides, dehydrated in ethanol, cleared in xylene, and mounted. The sections were observed and photographed by Olympus BX51.3 ImmunofluorescenceSpinal cord fixed in 4% paraformaldehyde was cryoprotected in a 30% sucrose/PBS solution for 12 h. Buried in OCT embedding medium, the tissues were frozen and sliced into 20μm sections using a Leica CM1850 Cryostat. Similar to immunohistochemistry, the sections were rinsed, perforated, blocked and incubated with anti-GFP, anti-Neu N, anti-GFAP, anti-Iba1, anti-APC antibodies, then incubated with corresponding fluorescence-labelled secondary antibodies for 1h at room temperature. After rinsing with PBS, sections were mounted with anti-fade solution and analyzed by confocal microscopy(Olympus FV1000).4 Western blottingAfter anaesthesia, lumbar, cervical spinal cord, brainstem, cerebellum, hippocampus, cortex and olfactory bulb were dissected out, rapidly frozen in liquid nitrogen and stored at-80℃ for western blot analysis. Whole tissue extracts were prepared using a total protein extraction kit following the manufacturer’s instruction and quantified using the BCA method. Thirty micrograms of protein from each sample was run on 10% SDS-PAGE gels, and blotted onto PVDF membranes. After the membranes were blocked with nonfat milk, primary antibodies were added respectively and the membranes were incubated overnight at 4 ℃. The next day, the membranes were rinsed, incubated with fluorescence-labeled secondary antibody. After incubation for 1 h at room temperature, the membranes were rinsed and the bands of interest were detected using an Odyssey Infrared Imaging System(LI-COR, Lincoln, NE) and statistically analyzed.5 Statistical analysesThe experimental data was demonstrated as mean± standard deviation( x ±s). Statistical analyses of protein expression in different groups were performed using one-way ANOVA with SPSS 13.0 statistical software. Differences were considered significant at P < 0.05.Results:1 Five-score evaluating system was established according to the transient weakness after intrathecal injection in mice. Score 1, minor weakness of hind limbs. Socre 2, moderate weakness of hind limbs with gait abnormality. Score 3, paralysis of hind limbs. Score 4, paralysis of hind limbs and weakness of fore limbs. Score 5, paralysis of all four limbs with breath difficulty. Mice scored 4-5 were successful. Fourteen out of 18 mice were successful and the successful rate was 77.8%.2 In the PBS-injected control mice, no GFP immunoreactivity was found in the spinal cord. r AAV9-injected mice showed different extent of GFP immunoreactivity in the spinal cord, which was correlated with the scores of limb weakness. The mice with scores of 4-5 showed obvious higher GFP expression in both lumbar and cervical spinal cord than those with scores under 3. Therefore, the extent of limb weakness after intrathecal injection can predict the transduction effect in the CNS three weeks later.3 The full length of the spinal cord was well transduced after intrathecal delivery of r AAV9 in mice. Particularly notable were the strong GFP-positive areas including the ventral and dorsal horns, the ventral effluent motor axons and dorsal affluent sensory axons. There was scattered transgene expression in brainstem, cerebellum and forebrain with varied extent. GFP positive cells were mainly glial cells, spinal cord anterior horn neurons, purkinje cells, cortical and hippocampus neurons were also seen expressing the transgene.4 Double immunofluorescence staining showed GFP expression in neurons and glial cells including astrocytes, oligodendrocytes, and microglia.5 Western blot showed that lumbar spinal cord had the highest GFP expression, then cervical spinal cord, olfactory bulb, hippocampus, and cerebral cortex, while brain stem and cerebellum showed similar relatively lower GFP expression.6 In the well r AAV9-injected mice, no astrogliosis or microgliosis was found in the lumbar spinal cord, and there was no significant difference compared with the control group.Conclusions:Intrathecal injection is simple and noninvasive, and can effectively predict CNS transduction efficiency in mice by adding lidocaine into the injection solute. r AAV9 delivered intrathecally to CSF transduced full length spinal cord and wide brain areas without inducing neuroinflammation, indicating potential application prospects in gene therapy for CNS disorders.