AAV2/5-mediated NGF gene delivery protects septal cholinergic neurons following axotomy

Nerve growth factor (NGF) therapy has been proposed to treat cognitive impairments in aged patients including those with Alzheimer's disease. Various viral vectors, including adeno-associated virus serotype 2 (AAV2), have been investigated for their ability to deliver NGF in brain. In this study, hybrid vectors (AAV2/5) consisting of the genome of recombinant AAV2 and the capsid of AAV serotype 5 were evaluated for their ability to deliver NGF and green fluorescent protein (GFP) genes into brain. Compared to AAV2, AAV2/5 consistently led to more septal neurons being transduced with GFP over a wider range of distribution. However, both types of vector provided similar levels of long-term (17 weeks) protection of septal cholinergic neurons from axotomy and led to similar levels of NGF accumulation in this region. These results demonstrate that rAAV-mediated NGF gene delivery is neuroprotective for an extended period of time, but that factors other than transduction efficiency appear to determine transgenic NGF expression in septum.     

Establishment of a recombinant adeno-associated virus expressing hVEGF_(165)

BACKGROUND: Because certain gene vectors could have deleterious effects in the central nervous system, the choice of a safe and effective vector system has become more important for gene therapy of nerve regeneration. OBJECTIVE: To construct a non-pathogenic, recombinant adeno-associated virus (AAV) simultaneously expressing human vascular endothelial growth factor 165 (hVEGF165) and green fluorescent protein (GFP). DESIGN, TIME AND SETTING: A randomized controlled experiment was performed at the Virology Laboratory of Shaanxi Provincial Center for Disease Control and Prevention between March and September 2007. MATERIALS: AAV helper-free system, AAV-293 packaging cell line, and AAV HT-1080 cells were purchased from Stratagene, USA. E. Coli DH5α was a stocked strain from Centers for Disease Control and Prevention of Shaanxi, China. Plasmid pUC18-hHVEGF165 was a gift from Zhibin Shi. METHODS: The hVEGF165 gene was amplified by PCR from pUC18-hHVEGF165 and inserted into plasmid pAAV-IRES-hrGFP to construct recombinant plasmid pAAV-hVEGF165-IRES-hrGFP. Subsequently pAAV-hVEGF165-IRES-hrGFP, pAAV-RC (the rep/cap-gene containing plasmid), and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hrGFP packaging through homologous recombination. The efficiency of AAV packaging was monitored under a fluorescent microscope, and the recombinant viral particles were harvested from infected AAV-293 cells, and further concentrated and purified. AAV HT-1080 cells were infected with the recombinant virus AAV-hVEGF165-IRES-hrGFP. MAIN OUTCOME MEASURES: Recombinant virus titer was measured by fluorescent cell counting, and infection efficiency was detected by Fluorescence Activated Cell Sorter (FACS) upon infecting AAV-HT1080 cells. The recombination with the exogenous gene was verified by PCR. RESULTS: The PCR amplified products were verified as hVEGF165 gene by DNA sequencing, and the recombinant pAAV-hVEGF165-IRES-GFP was confirmed by double digestion. The system provided a high packaging ratio of 95%, and the purified recombinant virus had a high titer of 5.5×1011 virus particles/mL. The AAV-HT1080 cells were infected at a ratio of 90.4%. The recombinant virus was confirmed by PCR to contain the exogenous hVEGF165 gene. CONCLUSION: The non-pathogenic rAAV-hVEGF165-GFP vector, carrying hVEGF165 and GFP reporter gene, was successfully constructed with a high titer and infection efficiency.     

Adeno-associated virus vector-mediated expression and constitutive secretion of galanin suppresses limbic seizure activity

Theoretically, gene therapy techniques offer an attractive alternative treatment option for intractable, focal epilepsies. Although logical gene therapy targets include excitatory and inhibitory receptors, variable viral vector tropism interjects an uncertainty as to the direction of change, seizure suppression, or seizure sensitization. To circumvent this therapeutic liability, adeno-associated virus (AAV) vectors have been constructed where the gene product is constitutively secreted from the transduced cell. Using AAV vectors, the fibronectin secretory signal sequence (FIB) was placed in front of the coding sequence for green fluorescent protein or the active portion of the neuroactive peptide galanin (GAL). Subsequent studies showed that these vectors supported expression and constitutive secretion of these gene products from transfected cells in vitro. More importantly, upon transduction in vivo, AAV-FIB-GAL vectors significantly attenuated focal seizure sensitivity, and this seizure attenuation could be controlled in vivo by using a tetracyclineregulated promoter. The expression and constitutive secretion of green fluorescent protein, or the expression of GAL alone, exerted no effect on focal seizure sensitivity. Moreover, unilateral infusion of the AAV-FIB-GAL vectors into the hippocampus prevented kainic acid-induced hilar cell death. With regard to limbic seizures, bilateral infusion of AAV-FIB-GAL vectors into the piriform cortex prevented both behavioral and localized electrographic seizure activity after the peripheral administration of kainic acid. Also, when rats were electrically kindled to class V seizure activity, subsequent infusion of AAV-FIB-GAL proved capable of significantly elevating the seizure initiation threshold. Thus, these studies clearly demonstrate the anti-seizure effectiveness of AAV vector-mediated expression and constitutive secretion of galanin.     

Adenoviral and adeno-associated viral vector mediated gene transfer in the guinea pig cochlea

Peripheral sensorineural hearing loss is a very common inner ear disorder affecting nearly 10% of the population. At present there is no cure for this disorder but gene therapy has been suggested as a potentially effective method for clinical treatment in the future. Thus we investigated the effectiveness of adenoviral (Ad) and adeno-associated viral (AAV) vectors to transduce the cochlea of guinea pigs. After direct injection into the basal turn of the cochlea, we found that both Ad and AAV vectors coding for the reporter genes lacZ or GFP readily transduced spiral ganglion cells. In addition, some transgene expression was detected in the stria vascularis after AAV-GFP injection. Gene expression persisted at least 8 weeks after viral vector injection. Present findings will help to develop future gene therapy protocols in the inner ear by using Ad and AAV coding for neurotrophins such as NT-3, BDNF, GDNF and VEGF.     

Systemic application of AAV vectors targeting GFAP-expressing astrocytes in Z-Q175-KI Huntington's disease mice

Huntington's disease (HD) affects both neurons and astrocytes. To target the latter and to ensure brain-wide transgene expression, adeno-associated viral (AAV) vectors can be administered intravenously, as AAV vectors cross the blood-brain barrier (BBB) and enable preferential transduction of astrocytes due to their close association with blood vessels. However, there is a possibility that the subclass of GFAP-expressing astrocytes performs a distinct role in HD and reacts differently to therapeutic measures than the rest of the astrocytes. The gfaABC1D promoter allows specific targeting of the GFAP-expressing astrocytes (~ 25% of S100β-expressing astrocytes). We have examined the expression of three different transgenes (GCaMP6f, Kir4.1 and GLT1) and tested the effects of the AAV serotypes 9 and rh8. The AAV vectors were injected into the tail vein of 1-year-old homozygous Z-Q175-KI HD mice and their wild-type (WT) littermates. At this age, HD mice exhibit motor symptoms, including pronounced hypokinesia and circling behaviour. The expression times ranged from 3 to 6 weeks. The target cell population was defined as the cells expressing S100β in addition to GFAP. Viewfields in the dorsal striatum and the overlaying cortex were evaluated and the transduction rate was defined as the percentage of target cells that expressed the reporter transgene (enhanced green fluorescent protein, EGFP, or Tomato). In all cases, the transduction rate was higher in the cortex than in the striatum. AAV9 was more efficient than AAVrh8. One of the injected constructs (AAV9-gfaABC1D-GLT1-Tomato) was tested for the first time. GLT1, the principal astrocytic glutamate transporter, is deficient in HD and therefore considered as a potential target for gene therapy. At a dose of 1.86 × 10¹¹ vector genome (vg) per animal, the fraction of GLT1-Tomato + cells in the striatum and the cortex amounted to 30% and 49%, respectively. In individual Tomato + HD astrocytes, treatment with the GLT1 vector increased the level of GLT1 immunofluorescence by 21% compared to the HD control. The described approach offers new and interesting opportunities to examine the pathophysiological consequences of brain-wide transgene expression in a specific astrocyte subpopulation.     

Comparison of AAV2 and AAV5 in gene transfer in the injured spinal cord of mice

Recombinant adeno-associated virus (AAV) vectors are promising tools for gene therapy. In spinal cord injury where extensive damage occurs, vectors with high diffusion and transduction abilities are required. We compared the diffusion capacity and transduction efficiency of AAV2 and AAV5 vectors using a mouse spinal cord injury model. Our study demonstrates that AAV5 is more effective than AAV2 for delivering genes into the injured spinal cord tissue. AAV5 diffused 6.9 mm from the injection site, transduced with an approximately two-fold increase in total cell number and yielded an approximately three-fold increase in gene expression in comparison with AAV2.     

Glial fibrillary acidic protein promoter determines transgene expression in satellite glial cells following intraganglionic adeno‐associated virus delivery in adult rats

Recombinant adeno‐associated viral (AAV)‐mediated therapeutic gene transfer to dorsal root ganglia (DRG) is an effective and safe tool for treating chronic pain. However, AAV with various constitutively active promoters leads to transgene expression predominantly to neurons, while glial cells are refractory to AAV transduction in the peripheral nervous system. The present study evaluated whether in vivo satellite glial cell (SGC) transduction in the DRG can be enhanced by the SGC‐specific GFAP promoter and by using shH10 and shH19, which are engineered capsid variants with Müller glia‐prone transduction. Titer‐matched AAV6 (as control), AAVshH10, and AAVshH19, all encoding the EGFP driven by the constitutively active CMV promoter, as well as AAV6‐EGFP and AAVshH10‐EGFP driven by a GFAP promoter (AAV6‐GFAP‐EGFP and AAVshH10‐GFAP‐EGFP), were injected into DRG of adult male rats. Neurotropism of gene expression was determined and compared by immunohistochemistry. Results showed that injection of AAV6‐ and AAVshH10‐GFAP‐EGFP induces robust EGFP expression selectively in SGCs, whereas injection of either AAVshH10‐CMV‐EGFP or AAVshH19‐CMV‐EGFP into DRG resulted in a similar in vivo transduction profile to AAV6‐CMV‐EGFP, all showing efficient transduction of sensory neurons without significant transduction of glial cell populations. Coinjection of AAV6‐CMV‐mCherry and AAV6‐GFAP‐EGFP induces transgene expression in neurons and SGCs separately. This report, together with our prior studies, demonstrates that the GFAP promoter rather than capsid tropism determines selective gene expression in SGCs following intraganglionic AAV delivery in adult rats. A dual AAV system, one with GFAP promoter and the other with CMV promoter, can efficiently express transgenes selectively in neurons versus SGCs.     

AAV8(gfp) preferentially targets large diameter dorsal root ganglion neurones after both intra-dorsal root ganglion and intrathecal injection

Adeno-associated viral vectors (AAV) are increasingly used to deliver therapeutic genes to the central nervous system (CNS) where they promote transgene expression in post mitotic neurones for long periods with little or no toxicity. In adult rat dorsal root ganglia (DRG), we investigated the cellular tropism of AAV8 containing the green fluorescent protein gene (gfp) after either intra-lumbar DRG or intrathecal injection and showed that transduced DRG neurones (DRGN) expressed GFP irrespective of the delivery route, while non-neuronal cells were GFP(-). After intra-DRG delivery of AAV8(gfp), the mean DRGN transduction rate was 11%, while intrathecal delivery transduced a mean of 1.5% DRGN. After intra-DRG injection, 2% of small DRGN (<30 μm in diameter) were GFP(+) compared with 32% of large DRGN (>60 μm in diameter). Axons of transduced DRGN were also GFP(+); no intra-spinal neurones were transduced. A small number of contralateral DRGN were transduced after intra-DRG injection, suggesting that AAV8 may diffuse from injected DRG into the spinal canal. Microglia and astrocytes were highly ramified with increased GFAP(+) immunoreactivity (i.e. activated) in the neuropil around GFP(+) DRG axon projections within the cord after intra-DRG injection. This study showed that after both intra-DRG and intrathecal delivery, strong preferential AAV8 tropism exists for large DRGN unassociated with cell death, but GFP(+) axons projecting in the spinal cord induced local glial activation. These results open up opportunities for targeted delivery of therapeutics such as neurotrophic factors to the injured spinal cord.     

Comparison of the efficacy of four viral vectors for transducing hypothalamic magnocellular neurosecretory neurons in the rat supraoptic nucleus

Since transgenes were first cloned into recombinant adenoviruses almost 30 years ago, a variety of viral vectors have become important tools in genetic research. Viruses adeptly transport genetic material into eukaryotic cells, and replacing all or part of the viral genome with genes of interest or silencing sequences creates a method of gene expression modulation in which the timing and location of manipulations can be specific. The hypothalamo-neurohypophyseal system (HNS), consisting of the paraventricular (PVN) and supraoptic (SON) nuclei in the hypothalamus, regulates fluid balance homeostasis and is highly plastic, yet tightly regulated by extracellular fluid (ECF) osmolality and volume. Its reversible plasticity and physiological relevance make it a good system for studying interactions between gene expression and physiology. Here, four viral vectors were compared for their ability to transduce magnocellular neurosecretory neurons (MNCs) of the SON in adult rats. The vectors included an adenovirus, a lentivirus (HIV) and two serotypes of adeno-associated viruses (AAV5 and AAV2). Though adenovirus and AAV2 vectors have previously been used to transduce SON neurons, HIV and AAV5 have not. All four vectors transduced MNCs, but the AAV vectors were the most effective, transducing large numbers of MNCs, with minimal or no glial transduction. The AAV vectors were injected using a convection enhanced delivery protocol to maximize dispersal through the tissue, resulting in the transduction of neurons throughout the anterior to posterior length of the SON (～1.5mm). AAV5, but not AAV2, showed some selectivity for SON neurons relative to those in the surrounding hypothalamus.     

Gene transfer into neurons from hippocampal slices: comparison of recombinant Semliki Forest Virus, adenovirus, adeno-associated virus, lentivirus, and measles virus

Viral vectors are useful for transferring genes into neurons. Here, we characterized recombinant Semliki Forest virus (SFV), adenovirus type 5 (Ad5), adeno-associated virus type 2 (AAV), lentivirus, and measles virus (MV) by their expression of green fluorescent protein (GFP) in rat hippocampal slice cultures. SFV infected more neurons (>90% of all GFP-positive cells) than AAV, lentivirus, and MV (71, 69, and 62%, respectively), whereas no infected neurons were identified with Ad5. AAV-mediated GFP expression was neuron-specific when the platelet-derived growth factor beta-chain promoter rather than cytomegalovirus promoter was used. Transgene expression occurred rapidly but transiently for SFV, increased slowly but remained stable with AAV and lentivirus, and was fast with MV. Resting membrane potential and conductance, action potentials, firing accommodation, and H-current appeared normal in infected CA1 pyramidal cells. Thus, SFV is useful for short-term and AAV and lentivirus for long-term transduction of hippocampal slices, while MV constitutes a novel vector.     

Adeno-associated viral vector-mediated gene transfer of brain-derived neurotrophic factor reverses atrophy of rubrospinal neurons following both acute and chronic spinal cord injury

Rubrospinal neurons (RSNs) undergo marked atrophy after cervical axotomy. This progressive atrophy may impair the regenerative capacity of RSNs in response to repair strategies that are targeted to promote rubrospinal tract regeneration. Here, we investigated whether we could achieve long-term rescue of RSNs from lesion-induced atrophy by adeno-associated viral (AAV) vector-mediated gene transfer of brain-derived neurotrophic factor (BDNF). We show for the first time that AAV vectors can be used for the persistent transduction of highly atrophic neurons in the red nucleus (RN) for up to 18 months after injury. Furthermore, BDNF gene transfer into the RN following spinal axotomy resulted in counteraction of atrophy in both the acute and chronic stage after injury. These novel findings demonstrate that a gene therapeutic approach can be used to reverse atrophy of lesioned CNS neurons for an extended period of time.     

Canine distemper virus persistence in demyelinating encephalitis by swift intracellular cell-to-cell spread in astrocytes is controlled by the viral attachment protein

The mechanism of viral persistence, the driving force behind the chronic progression of inflammatory demyelination in canine distemper virus (CDV) infection, is associated with non-cytolytic viral cell-to-cell spread. Here, we studied the molecular mechanisms of viral spread of a recombinant fluorescent protein-expressing virulent CDV in primary canine astrocyte cultures. Time-lapse video microscopy documented that CDV spread was very efficient using cell processes contacting remote target cells. Strikingly, CDV transmission to remote cells could occur in less than 6 h, suggesting that a complete viral cycle with production of extracellular free particles was not essential in enabling CDV to spread in glial cells. Titration experiments and electron microscopy confirmed a very low CDV particle production despite higher titers of membrane-associated viruses. Interestingly, confocal laser microscopy and lentivirus transduction indicated expression and functionality of the viral fusion machinery, consisting of the viral fusion (F) and attachment (H) glycoproteins, at the cell surface. Importantly, using a single-cycle infectious recombinant H-knockout, H-complemented virus, we demonstrated that H, and thus potentially the viral fusion complex, was necessary to enable CDV spread. Furthermore, since we could not detect CD150/SLAM expression in brain cells, the presence of a yet non-identified glial receptor for CDV was suggested. Altogether, our findings indicate that persistence in CDV infection results from intracellular cell-to-cell transmission requiring the CDV-H protein. Viral transfer, happening selectively at the tip of astrocytic processes, may help the virus to cover long distances in the astroglial network, “outrunning” the host’s immune response in demyelinating plaques, thus continuously eliciting new lesions.     

Neuroprotective adeno-associated virus Bcl-xL gene transfer in models of motor neuron disease

Recent work implicates excitotoxicity-induced apoptosis as the mechanism triggering motor neuron death in amyotrophic lateral sclerosis (ALS). Our laboratory has previously utilized glutamate excitotoxicity in vitro to study this process. The present experiment tests whether overexpression of the gene for Bcl-xL can inhibit excitotoxicity in this model system. To track Bcl-xL expression, the gene for green fluorescent protein (GFP) was inserted in-frame, upstream of the Bcl-xL gene. The GFP-Bcl-xL gene was then cloned into an adeno-associated viral (AAV2) vector. GFP expression in both SH-SY5Y and embryonic day 15 (E15) motor neurons (MNs) peaked 48 hours after infection. Bcl-xL expression in SH-SY5Y cells significantly reduced terminal deoxy-UTP nick-end labeling (TUNEL)-positive cells and maintained cell density after glutamate exposure. Similarly, Bcl-xL expression inhibited the development of TUNEL staining in E15 MNs and supported cell density after glutamate exposure. These findings suggest that AAV-mediated expression of genes for antiapoptotic proteins may provide a means for ALS gene therapy.     

Intraneural convection enhanced delivery of AAVrh20 for targeting primary sensory neurons

Gene therapy using adeno-associated virus (AAV) is an attractive strategy to treat disorders of the peripheral nervous system (PNS), such as chronic pain or peripheral neuropathies. Although intrathecal (IT) administration of AAV has been the standard in the field for targeting the PNS, it lacks anatomical specificity and results in wide rostro-caudal distribution of the vector. An alternative approach is to deliver AAV directly to the peripheral nerve axon. The present study employed convection-enhanced delivery (CED) of a novel AAV serotype, AAVrh20, expressing enhanced green fluorescent protein (EGFP) into rat sciatic nerve investigating its efficacy, anatomical selectivity, and safety, compared to the IT route. Intraneural CED resulted in transduction confined to the ipsilateral L4 and L5 DRG while IT administration led to promiscuous DRG transduction encompassing the entire lumbar region bilaterally. The transduction rate for intraneural AAV administration was similar to IT delivery (24% for L4 and 31.5% for L5 DRG versus 50% for L4 and 19.5% for L5 DRG). The use of hyperosmotic diluent did not further improve the transduction efficiency. AAVrh20 was superior to reference serotypes previously described to be most active for each route. Intraneural CED of AAV was associated with transient allodynia that resolved spontaneously. These findings establish intraneural CED as an alternative to IT administration for AAV mediated gene transfer to the PNS and, based on a reference rodent model, suggest AAVrh20 as a superior serotype for targeting the PNS.     

Phenotype correction of hemophilia A mice with adeno-associated virus vectors carrying the B domain-deleted canine factor VIII gene

Adeno-associated virus (AAV) vectors carrying the B domain-deleted canine FVIII (BDD cFVIII) gene utilizing the beta-actin minimum promoter (167b) pseudotyped with serotype 1 (AAV1-beta-actin-cFVIII) and serotype 8 (AAV8-beta-actin-cFVIII) were developed to express cFVIII in hemophilia A mice. FVIII clotting activities measured by the APTT method increased in hemophilia A mice with intramuscular injection of AAV1-beta-actin-cFVIII in a dose-dependent manner. Therapeutic FVIII levels (2.9+/-1.0%) in hemophilia A mice with the AAV1-beta-actin-cFVIII dose of 1 x 10(12) gc/body were achieved, suggesting partial correction of the phenotype with AAV1-beta-actin-cFVIII vectors. FVIII clotting activity levels in hemophilia A mice with intravenous injection of AAV8-beta-actin-cFVIII also were increased dose-dependently, achieving therapeutic FVIII levels (5-90%) in hemophilia A mice with the AAV8-beta-actin-cFVIII doses of 1-3 x 10(11) gc/body and supernormal FVIII levels (180-670%) in hemophilia A mice with the AAV8-beta-actin-cFVIII dose of 1 x 10(12) gc/body. Transduction of the liver with AAV8-beta-actin-cFVIII is superior to transduction of skeletal muscles with AAV1cFVIII regarding the FVIII production and antibody formation. These data suggested that both AAV1 and AAV8 vectors carrying the FVIII gene utilizing a minimum promoter have a potential for hemophilia A gene therapy.     

Lentiviral Vector Mediates Exogenous Gene Expression in Adult Rat DRG Following Peripheral Nerve Remote Delivery

The primary sensory neurons with cell bodies in the dorsal root ganglion (DRG) have been extensively used as models in neurobiology and provide a useful model to study the mechanism of neural regeneration. Therefore, efficient and stable gene delivery to these postmitotic cells has significant therapeutic potential. Various studies involving the viral vector systems capable of neuronal transduction have been extensively evaluated in the cultured DRG neurons by adeno-associated virus. In the present study, we investigated the transduction performance of the lentiviral vector that mediates the catalytic subunit of protein kinase A (PKAc) and green fluorescent protein (GFP) expression in the DRG by sciatic nerve retrograde transport and tested whether PKAc expression in the DRG could inhibit the activation of RhoA after spinal cord injury. Five days after sciatic nerve remote delivery of lentiviral vector (LV)/PKAc-internal ribosome entry site (IRES)-GFP or LV/GFP, the L4–L6 DRGs were dissected for primary culture or immunostaining to observe the exogenous gene expression, or transecting the dorsal part of lumbar enlargement was performed, and 16 h later, the function of the exogenous gene was tested by RhoA pull-down analysis. The results showed that the lentiviral vector could mediate exogenous gene PKAc expression in the DRG and then inhibit spinal cord injury-induced RhoA activation by remote delivery of LV/PKAc-IRES-GFP through the sciatic nerve.     

小鼠HMGB1启动子红色荧光蛋白报告基因载体的构建及在真核细胞中的表达

目的 构建小鼠高迁移率族蛋白B1(HMGB1)启动子驱动的红色荧光蛋白报告基因载体,为研究细胞内HMGB1的基因表达调控和相关的信号转导机制提供重要工具.方法 采用基因重组技术构建含HMGB1启动子区的红色荧光蛋白报告基因载体pDsRed1-1-HMGB1p;脂质体法瞬时转染NIH3T3细胞.观察HMGB1启动子存正常情况下以及肿瘤坏死因子-α(TNF-α)刺激后的细胞内活性.结果 酶切鉴定和DNA测序证明所构建的红色荧光蛋白报告基凼载体正确:该载体在NIH3T3细胞内静息状态呈低水平表达,经TNF-α刺激后,观察到表达高亮度红色荧光的细胞明显增多.结论所构建的HMGB1启动子红色荧光蛋白报告基因载体正确,具有正常启动蛋白表达的活性,可有效用于HMGB1基因表达信号调控机制的研究.     

Gene therapy for Parkinson's disease: studies in animal models]

Recent developments in viral vectors capable of providing high levels of long-term transgene expression in the brain have led to the pursuit of two strategies in gene therapy for the treatment of Parkinson's disease (PD). One is the local production of dopamine in the striatum achieved by inducing the expression of dopamine-synthesizing enzymes. Three enzymes are necessary for efficient dopamine synthesis: tyrosine hydroxylase (TH) converts tyrosine to L-DOPA, aromatic L-amino acid decarboxylase (AADC) then converts L-DOPA to dopamine, and guanosine triphosphate cyclohydrolase I (GCH) is the rate-limiting enzyme for the synthesis of TH co-factor tetrahydrobiopterine. We have previously demonstrated that transduction with separate adeno-associated virus (AAV) vectors expressing TH, AADC, and GCH is effective in reducing motor abnormalities in 6-hydroxydopamine-lesioned rats and in MPTP-treated monkeys. Behavioral recovery persisted for at least 18 months after intrastriatal injection in parkinsonian rats. In MPTP monkeys, the amelioration of motor abnormalities was remarkable on the contralateral side, accompanied by robust transgene expression and elevated dopamine synthesis in the AAV-injected putamen. The second strategy entails the expression of neurotrophic factors or brain vesicular monoamine transporter in the striatum or the substantia nigra to slow the degeneration of dopamine neurons. Gene therapy using viral vectors offers a promising approach in the treatment of PD patients.     

Visualization of spinal afferent innervation in the mouse colon by AAV8‐mediated GFP expression

Background Primary afferent neurons whose cell bodies reside in thoracolumbar and lumbosacral dorsal root ganglia (DRG) innervate colon and transmit sensory signals from colon to spinal cord under normal conditions and conditions of visceral hypersensitivity. Histologically, these extrinsic afferents cannot be differentiated from intrinsic fibers of enteric neurons because all known markers label neurons of both populations. Adeno‐associated virus (AAV) vectors are capable of transducing DRG neurons after intrathecal administration. We hypothesized that AAV‐driven overexpression of green fluorescent protein (GFP) in DRG would enable visualization of extrinsic spinal afferents in colon separately from enteric neurons. Methods Recombinant AAV serotype 8 (rAAV8) vector carrying the GFP gene was delivered via direct lumbar puncture. Green fluorescent protein labeling in DRG and colon was examined using immunohistochemistry. Key Results Analysis of colon from rAAV8‐GFP‐treated mice demonstrated GFP‐immunoreactivity (GFP‐ir) within mesenteric nerves, smooth muscle layers, myenteric plexus, submucosa, and mucosa, but not in cell bodies of enteric neurons. Notably, GFP‐ir colocalized with CGRP and TRPV1 in mucosa, myenteric plexus, and globular‐like clusters surrounding nuclei within myenteric ganglia. In addition, GFP‐positive fibers were observed in close association with blood vessels of mucosa and submucosa. Analysis of GFP‐ir in thoracolumbar and lumbosacral DRG revealed that levels of expression in colon and L6 DRG appeared to be related. Conclusions & Inferences These results demonstrate the feasibility of gene transfer to mouse colonic spinal sensory neurons using intrathecal delivery of AAV vectors and the utility of this approach for histological analysis of spinal afferent nerve fibers within colon.