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Recombinant adeno-associated viral (AAV) vectors are one of the most promising therapeutic delivery systems for gene therapy to the central nervous system (CNS). Preclinical testing of novel gene therapies requires the careful design and production of AAV vectors and their successful application in a model of CNS injury. One major limitation of AAV vectors is their limited packaging capacity (<5 kb) making the co-expression of two genes (e.g., from two promoters) difficult. An Internal Ribosomal Entry Sequence (IRES) has been used to express two genes: However, the second transgene is often expressed at lower levels than the first. In addition to this, achieving high levels of transduction in the CNS can be challenging. In this chapter we describe the cloning of a bicistronic AAV vector that uses the foot and mouth disease virus 2A sequence to efficiently express two genes from a single promoter. Bicistronic expression of a therapeutic gene and a reporter gene is desirable so that the axons from transduced neurons can be tracked and, after CNS injury, the amount of axonal sprouting or regeneration quantified. We go on to describe how to perform a pyramidotomy model of CNS injury and the injection of AAV vectors into the sensorimotor cortex to provide efficient transduction and bicistronic gene expression in cortical neurons such that transduced axons are detectable in the dorsal columns of the spinal cord.
Following an injury to the central nervous system (CNS) neurons undergo collateral sprouting but very limited long distance regeneration which results in their failure to form functional connections with their original targets. This is due in part to the reduced intrinsic growth state of mature CNS neurons, which is characterised by their failure to express regeneration associated genes (RAGs) after an injury (1–5). Steady advancements in molecular neurobiology have elevated gene therapy into a promising therapeutic strategy for increasing the expression of RAGs and improving regeneration after a CNS injury (6–9).
To test a potential gene therapy an in vivo model of CNS injury is required. The corticospinal tract (CST) is present in all mammals and originates from the pyramidal corticospinal neurons (CSNs) in layer V of the cerebral cortex that project through the brainstem to the spinal cord. The CST fibres descend through the ventral brainstem in the medullary pyramids after which the main component of the CST decussates at the spinomedullary junction to form the dorsal CST which runs in the ventral portion of the contralateral dorsal funiculus in the adult rat (10). The CST is primarily a descending motor pathway that controls locomotion, posture and voluntary skilled movements (11). Following injury to the rodent CST, persistent deficits in sensorimotor function are observed and restorative strategies are often assessed using behavioural tests of sensorimotor and skilled motor function (11–14). The CST can be lesioned by performing a unilateral pyramidotomy (i.e. one of the medullary pyramids is cut) (15). This leads to the anterograde degeneration of the injured axons and functional deficits on the contralateral side of the body. These deficits can be overcome by enhancing the growth state of the uninjured CSNs, allowing them to sprout their axons across the midline and reinnervate the area denervated by the lesion. The pyramidotomy model has been extensively used to assess potential therapies to enhance neuronal regeneration, sprouting and functional recovery following CNS injury (8, 16). However, efficient, wide-spread transduction of the CSNs in the cortex is challenging and has only been achieved when high titres (1x1014 Genome Copies (GC)/ml) and slow infusion rates were used (6, 8, 17). Here we outline a procedure to inject AAVs into the cortex to transduce CSNs and then perform a pyramidotomy injury.
Recombinant adeno-associated viral (AAV) vectors represent one of the most attractive gene delivery systems for the CNS due to their ability to efficiently transduce neurons and to provide long-term expression with only a minimal immune response (18, 19). AAV vectors have one of the best characterised safety profiles and have been taken to clinical trial for a number of neurodegenerative diseases (20, 21). In addition to this, Glybera (an AAV vector encoding lipoprotein lipase) has recently been approved for use in the EU to reduce incidence of pancreatitis in people with inherited lipoprotein lipase deficiency (22) and paves the way for further AAV based gene therapies for other conditions. Each AAV serotype expresses different capsid proteins that determine which receptors the AAV vector can bind to for cell entry and therefore establishes the AAV vector’s tropism (23). Pseudotyping AAV2 genome with the capsid from different AAV serotypes can generate vectors with different tissue and cellular tropisms, which can improve the efficiency and pattern of transduction in specific regions of the CNS (24–26). We have previously shown that AAV1 is the optimal serotype for transducing CSNs in adult rats (17) although other AAVs also work well; AAV5 (27–29) and AAV8 (6, 28, 30). Viral vector mediated over-expression of pro-regenerative genes, including retinoic acid receptor, beta (RARbeta2), neuronal calcium sensor 1 (NCS1) and Kruppel-like factor 7 (KLF-7) have been shown to enhance the intrinsic growth state of CSNs and facilitate axonal regeneration after injury (6–9). However, these studies have either used viral vectors with larger insert capacities such as lentiviral vectors or used two separate AAV vectors; one encoding a RAG and the other a reported gene. It would therefore be very valuable to have an AAV which can co-express a pro-regenerative gene together with a fluorescent reporter so that transduced axons can be distinguished from axons that are not transduced: this would increase our ability to identify useful pro-regenerative therapies.
A disadvantage of AAV vectors is their relatively low insert capacity which can be an issue if two genes are required to be expressed (e.g., from two promoters). One partial solution is to use a single promoter which drives two genes linked by an internal ribosomal entry site (IRES) (31, 32): typically the RAG would be expressed immediately downstream of the promoter whilst the fluorescent reporter gene is expressed downstream of the IRES. Although this works reasonably well, the IRES-dependent second gene is usually expressed at a significantly lower level, estimated at 10-fold less than the cap-dependent first gene (33, 34). To date we know of no reports where transduction of supraspinal neuronal cell bodies with such an AAV results in detectable labelling of their axons in the spinal cord.
This problem can be mitigated by using the 2A peptide from the Foot and Mouth Disease Virus (35). The 2A peptide sequence is only 24 amino acids long and can mediate efficient bicistronic expression of two gene products from a single promoter by undergoing ribosomal skipping (referred to as "self-cleavage") during protein translation. Theoretically, the two gene products are expressed in a 1:1 ratio and in practice each gene product is expressed at a high level. The 2A and 2A-like bicistronic systems have been shown to be highly effective in the CNS where high expression levels of both genes have been demonstrated (6, 34).
The psubCMV-2A-WPRE plasmid (Figure 1A) was a kind gift from Dr. Hansruedi Bueler, University of Kentucky. The plasmid contains a cytomegalovirus (CMV) promoter (to drive expression of the bicistron), the Foot-and-mouth disease virus (FMDV) 2A sequence and a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE). The 2A sequence is immediately flanked by an upstream (i.e., 5’) NheI restriction site and a downstream (i.e., 3’) MluI restriction site (Figure 1B).
NheI and MluI, NEBuffer 4 and BSA (NEB, UK). Alkaline phosphatase (Calf intestinal) (NEB, UK).
Vent DNA polymerase (NEB, UK), 10X Vent DNA polymerase buffer, DMSO, 10 mM Nucleotide mix (Promega, UK), 100 mM MgSO4, 100 μM forward and reverse primers, 50 ng plasmids containing mCherry or EGFP, PCR-grade nucleotide free H2O.
0.5 mg/kg Domitor (medetomidine hydrochloride) and 100 mg/kg Vetalar (ketamine hydrochloride) or with a mixture of Isoflurane (1.5 %) and O2 (1 L/min).
5 mg/kg Carprofen was administered subcutaneously for post-operative analgesia.
400 mg/kg Lethabarb (sodium pentobarbital).
Our goal was to generate a series of plasmids with the generic structure (AAV-RAG-2A-EGFP). First, we cloned EGFP downstream of 2A and then we cloned mCherry upstream of 2A. The resulting plasmid (AAV-mCherry-2A-EGFP-WPRE) (Figure 1C) has been used in many of our experiments as a negative control (because mCherry is in place of a given RAG). It also enabled us to check easily that, indeed, both genes are expressed (Figures 2 and and3).3). Subsequently, we have cloned more than 10 different pro-regenerative transgenes in place of mCherry.
Forward and reverse primers were designed for EGFP (Table 1) and mCherry (Table 2) to ensure that the coding region of the plasmid produced one single open reading frame encoding all of the following: a start codon, the upstream cDNA (mCherry in this case), the 2A sequence, and the downstream cDNA (EGFP in this case), and a stop codon.
For cloning EGFP downstream of the 2A sequence, the forward primer was designed to contain a hexamer of GC-repeats (blue) for stability of the PCR product and efficient restriction enzyme cleavage, an MluI restriction site (yellow), and the first 28 bases of the EGFP cDNA excluding the ATG start codon. The reverse primer contained a hexamer of GC-repeats (blue), an MluI restriction site (yellow), and the reverse complement of the last 28 bases of the EGFP cDNA (green) including the stop codon (underlined) to ensure that translation ends after the synthesis of the bicistron.
For cloning mCherry upstream of the 2A, the forward primer was designed to contain a hexamer of GC-repeats (blue) for stability of the PCR product and efficient restriction enzyme cleavage, a NheI restriction site (pink), and the first 28 bases of the mCherry cDNA (red) including the start codon (ATG; underlined). The reverse primer contained the GC-repeat hexamer (blue), a NheI restriction site (pink), and the reverse complement of the last 28 bases of the mCherry cDNA with the stop codon excluded (red).
Primers were synthesised at 50 nmoles with desalting but no other purification (Sigma, UK). We always check the resulting AAV plasmids by DNA sequencing to ensure that sequences are correct.
Sequencing primer used to check the success of cloning a gene upstream of 2A: 5'-AGC-TGC-GGA-ATT-GTA-CCC-GC-3'
Sequencing primer used to check the success of cloning a gene downstream of 2A: 5'-AAG-GCA-TTA-AAG-CAG-CGT-ATC-CAC-A-3'
The reagents included in our standard PCR were:
|Volume (µl)||Final concentration|
|DNA template||1||Determined by user; we used 50 ng|
|Forward primer (100 µM)||1||1 µM|
|Reverse primer (100 µM)||1||1 µM|
|ThermoPol Reaction Buffer (10x)||10||1x|
|Vent polymerase (2 units/µL)||1||2 units|
|dNTPs (pool of 10 mM of each)||2||200 µM|
|MgSO4 (100 mM)||4||4 mM|
|Nuclease-free water||To a final volume of 100 µl|
AAV vectors were generated by the Miami Project Viral Vector Core using fast protein liquid chromatography (FPLC)-based purification and pseudotyped with AAV capsid serotype 1. AAV vectors were suspended in HBSS at 2.9x1013 GC/ml.
Female Lister Hooded rats weighing 200-220 g (Charles River, UK) were used for these experiments. Rats were maintained under standard animal care conditions (12:12 hr light/dark cycle), with food and water ad libitum. All procedures were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and approved by the local veterinarian and ethical committee.
Holes were drilled through the skull above the sensorimotor cortex using coordinates reported in a microstimulation mapping study relative to Bregma, midline and the brain surface (36, 37) (see methods section for coordinates).
Stereotaxic frame for rats (World precision instruments, FL, USA) which allowed the attachment of a microdrill (Power performance, UK) or ultra microsyringe pump with Micro4 control panel (World Precision Instruments, FL, USA).
Eye lubricant (Viscotears, Germany), alcohol-based permanent marker, chlorhexidine disinfectant, sterile cotton buds, small bulldog clamps (World Precision Instruments # 14118), blunted scissors (Fine Science Tools, Fine Scissors – Tough Cut 14058-09), toothed forceps (Fine Science Tools, 11019-12), Long-toothed Alm retractors (Fine Science Tools, 17009-07), fine Dumont forceps (Fine Science Tools, 11251-10), dental hand drill, 0.7 mm drill bits (Fine Science Tools, 19007-07), Vannas Spring Scissors (Fine Science Tools, 15000-03), gelfoam (Equimedical, Gelatin Absorbable Haemostatic Sterile EQU705001), 3.0 Vicryl sutures (Ethicon), scalpel and #10 scalpel blades, needle holder, 27-G needles, 1 ml disposable syringes (for intraperitoneal (i.p.) injection of the anaesthetic), 10 μl Hamilton syringe (Hamilton, Reno, NV, USA), low magnification stereomicroscope (2-20x).
4% paraformaldehyde in phosphate-buffered saline (PBS) pH 7 and cryoprotectant (30% sucrose in PBS).
10% porcine gelatine in ddH2O or cryoprotectant embedding medium OCT. A freezing microtome (Leitz, Wetzlar, Germany) or cryostat (Bright, UK) was used to cut the tissue.
Confocal microscope (Carl Zeiss LSM 710)
|1 cycle||Holding||95||2 minutes|
|25 cycles||Denaturation||95||30 seconds|
|1 cycle||Final extension||73||11 minutes|
|DNA (Up to 1 µg)||1 µl|
|10x Buffer 4||2 µl|
|100x BSA||0.2 µl|
|Restriction enzyme (NheI or MluI)||0.5 µl (2-10 units)|
|Nuclease-free water||To a final volume of 20 µl|
In this section we describe methods for the injection of AAVs into the cortex of adult rats. We also describe methods for performing a pyramidotomy. Depending on the goal of the experiment, AAVs can be injected prior or subsequent to the CNS injury.
We would like to thank Dr. Vance Lemmon and the Miami Vector Core for production of the AAV vectors. We would also like to acknowledge support from a Research Councils UK Academic Fellowship (L.M.), the British Pharmacological Society’s Integrative Pharmacology Fund (L.M), Friends of Guy’s Hospital Research Grants (L.M.) and a grant from the Henry Smith Charity (L.M. and T.H.).