Patrick N. Anderson

Effective gene therapy with nonintegrating lentiviral vectors

Retroviral and lentiviral vector integration into host-cell chromosomes carries with it a finite chance of causing insertional mutagenesis. This risk has been highlighted by the induction of malignancy in mouse models, and development of lymphoproliferative disease in three individuals with severe combined immunodeficiency–X1 (refs. 2,3). Therefore, a key challenge for clinical therapies based on retroviral vectors is to achieve stable transgene expression while minimizing insertional mutagenesis. Recent in vitro studies have shown that integration-deficient lentiviral vectors can mediate stable transduction. With similar vectors, we now show efficient and sustained transgene expression in vivo in rodent ocular and brain tissues. We also show substantial rescue of clinically relevant rodent models of retinal degeneration. Therefore, the high efficiency of gene transfer and expression mediated by lentiviruses can be harnessed in vivo without a requirement for vector integration. For therapeutic application to postmitotic tissues, this system substantially reduces the risk of insertional mutagenesis.

The Nogo receptor, its ligands and axonal regeneration in the spinal cord; A review

At least three proteins present in CNS myelin, Nogo, MAG and OMgp are capable of causing growth cone collapse and inhibiting neurite outgrowth in vitro. Surprisingly, Nogo and OMgp are also strongly expressed by many neurons (including neocortical projection cells). Nogo expression is increased by some cells at the borders of CNS lesion sites and by cells in injured peripheral nerves, but Nogo and CNS myelin are largely absent from spinal cord injury sites, which are none the less strongly inhibitory to axonal regeneration. Nogo is found on growing axons during development, suggesting possible functions for neuronal Nogo in axon guidance. Although Nogo, MAG and OMgp lack sequence homologies, they all bind to the Nogo receptor (NgR), a GPI-linked cell surface molecule which, in turn, binds p75 to activate RhoA. NgR is strongly expressed by cerebral cortical neurons but many other neurons express NgR weakly or not at all. Some neurons, such as DRG cells, respond to Nogo and CNS myelin in vitro although they express little or no NgR in vivo which, with other data, indicates that other receptors are available for NgR ligands. NgR expression is unaffected by injury to the nervous system, and there is no clear correlation between NgR expression by neurons and lack of regenerative ability. In the injured spinal cord, interactions between NgR and its ligands are most likely to be important for limiting regeneration of corticospinal and some other descending tracts; other receptors may be more important for ascending tracts. Antibodies to Nogo, mainly the poorly-characterised IN-1 or its derivatives, have been shown to enhance recovery from partial transections of the spinal cord. They induce considerable plasticity from the axons of corticospinal neurons, including sprouting across the midline and, to a limited extent, regeneration around the lesion. Regeneration of corticospinal axons induced by Nogo antibodies has not yet been demonstrated after complete transections or contusion injuries of the spinal cord. It is not clear whether antibodies against Nogo act on oligodendrocytes/myelin or by binding to neuronal Nogo, or whether they can stimulate regeneration of ascending axons in the spinal cord, most of which express little or no NgR. Despite these uncertainties, however, NgR and its ligands offer important new targets for enhancing plasticity and regeneration in the nervous system.

Peripheral nerve injury fails to induce growth of lesioned ascending dorsal column axons into spinal cord scar tissue expressing the axon repellent Semaphorin3A

We have investigated the hypothesis that the chemorepellent Semaphorin3A may be involved in the failure of axonal regeneration after injury to the ascending dorsal columns of adult rats. Following transection of the thoracic dorsal columns, fibroblasts in the dorsolateral parts of the lesion site showed robust expression of Semaphorin3A mRNA. In addition, dorsal root ganglion (DRG) neurons with projections through the dorsal columns to the injury site persistently expressed both Semaphorin3A receptor components, neuropilin‐1 and plexin‐A1. These ascending DRG collaterals failed to invade scar regions occupied by Semaphorin3A‐positive fibroblasts, even in animals which had received conditioning lesions of the sciatic nerve to enhance regeneration. Other axon populations in the dorsal spinal cord were similarly unable to penetrate Semaphorin3A‐positive scar tissue. These data suggest that Semaphorin3A may create an exclusion zone for regenerating dorsal column fibres and that enhancing the intrinsic regenerative response of DRG neurons has only limited effects on axonal regrowth. Tenascin‐C and chondroitin sulphate proteoglycans were also detected at the injury site, which was largely devoid of central nervous system (CNS) myelin, showing that several classes of inhibitory factors, including semaphorins, with only partially overlapping spatial and temporal patterns of expression are in a position to participate in preventing regenerative axonal growth in the injured dorsal columns. Interestingly, conditioning nerve injuries enabled numerous ascending DRG axons to regrow across areas of strong tenascin‐C and chondroitin sulphate proteoglycan expression, while areas containing Semaphorin3A and CNS myelin were selectively avoided by (pre)primed axonal sprouts.

The Effects of FK506 on Dorsal Column Axons Following Spinal Cord Injury in Adult Rats: Neuroprotection and Local Regeneration

There is considerable evidence that immunophilin ligands can promote the regeneration of axons in peripheral nerves and act as neuroprotective agents in the CNS. We have examined the effects of FK506 and GPI 1046 on the responses to partial transection of ascending spinal dorsal column axons at T9, in some cases combined with crush of one sciatic nerve. FK506 (0.5 or 2.0 mg/kg) and GPI 1046 (10 or 40 mg/kg) was administered subcutaneously immediately after surgery and five times a week thereafter. Some animals received methylprednisolone (MP) (two subcutaneous doses of 30 mg/kg) in addition to, or instead of, FK506. After survival times of 1-12 weeks, dorsal column axons were labeled transganglionically with cholera toxin B-HRP. There was massive axonal sprouting at the lesion sites in animals with sciatic nerve injury and immunophilin ligand treatment. In FK506-treated animals a few severed sensory axons regenerated for up to 10 mm rostral to the lesion. Of greater significance, 30% of 71 FK506-treated animals had spared axons in the dorsal column, extending to the nucleus gracilis, versus 8% of 50 control animals (P < 0.05), showing that FK506 reduces the likelihood of axonal destruction due to secondary injury. A combination of FK506 and MP afforded greater protection than MP alone (P < 0.05), but axonal survival was not affected by sciatic nerve crush, dose of FK506, or survival time after injury. GPI 1046 (n = 11) did not promote axonal survival. Thus FK506 protects axons from secondary injury following spinal cord trauma, and in this experimental model, its neuroprotective effect is greater than that of MP.

Corticospinal neurons up‐regulate a range of growth‐associated genes following intracortical, but not spinal, axotomy

The failure of some CNS neurons to up‐regulate growth‐associated genes following axotomy may contribute to their failure to regenerate axons. We have studied gene expression in rat corticospinal neurons following either proximal (intracortical) or distal (spinal) axotomy. Corticospinal neurons were retrogradely labelled with cholera toxin subunit B prior to intracortical lesions or concomitantly with spinal lesions. Alternate sections of forebrain were immunoreacted for cholera toxin subunit B or processed for mRNA in situ hybridization for ATF3, c‐jun, GAP‐43, CAP‐23, SCG10, L1, CHL1 or krox‐24, each of which has been associated with axotomy or axon regeneration in other neurons. Seven days after intracortical axotomy, ATF3, c‐jun, GAP‐43, SCG10, L1 and CHL1, but not CAP‐23 or krox‐24, were up‐regulated by layer V pyramidal neurons, including identified corticospinal neurons. The maximum distance between the lesion and the neuronal cell bodies that up‐regulated genes varied between 300 and 500 µm. However, distal axotomy failed to elicit changes in gene expression in corticospinal neurons. No change in expression of any molecule was seen in the neocortex 1 or 7 days after corticospinal axotomy in the cervical spinal cord. The expression of GAP‐43, CAP‐23, L1, CHL1 and SCG10 was confirmed to be unaltered after this type of injury in identified retrogradely labelled corticospinal neurons. Thus, while corticospinal neuronal cell bodies fail to respond to spinal axotomy, these cells behave like regeneration‐competent neurons, up‐regulating a wide range of growth‐associated molecules if axotomized within the cerebral cortex.

Multiple Immediate-Early Gene-Deficient Herpes Simplex Virus Vectors Allowing Efficient Gene Delivery to Neurons in Culture and Widespread Gene Delivery to the Central Nervous System In Vivo

ABSTRACT Herpes simplex virus (HSV) has several potential advantages as a vector for delivering genes to the nervous system. The virus naturally infects and remains latent in neurons and has evolved the ability of highly efficient retrograde transport from the site of infection at the periphery to the site of latency in the spinal ganglia. HSV is a large virus, potentially allowing the insertion of multiple or very large transgenes. Furthermore, HSV does not integrate into the host chromosome, removing any potential for insertional activation or inactivation of cellular genes. However, the development of HSV vectors for the central nervous system that exploit these properties has been problematical. This has mainly been due to either vector toxicity or an inability to maintain transgene expression. Here we report the development of highly disabled versions of HSV-1 deleted for ICP27, ICP4, and ICP34.5/open reading frame P and with an inactivating mutation in VP16. These viruses express only minimal levels of any of the immediate-early genes in noncomplementing cells. Transgene expression is maintained for extended periods with promoter systems containing elements from the HSV latency-associated transcript promoter (J. A. Palmer et al., J. Virol. 74:5604–5618, 2000). Unlike less-disabled viruses, these vectors allow highly effective gene delivery both to neurons in culture and to the central nervous system in vivo. Gene delivery in vivo is further enhanced by the retrograde transport capabilities of HSV. Here the vector is efficiently transported from the site of inoculation to connected sites within the nervous system. This is demonstrated by gene delivery to both the striatum and substantia nigra following striatal inoculation; to the spinal cord, spinal ganglia, and brainstem following injection into the spinal cord; and to retinal ganglion neurons following injection into the superior colliculus and thalamus.

Regulation of Nogo and Nogo receptor during the development of the entorhino-hippocampal pathway and after adult hippocampal lesions.

Axonal regeneration in the adult CNS is limited by the presence of several inhibitory proteins associated with myelin. Nogo-A, a myelin-associated inhibitor, is responsible for axonal outgrowth inhibition in vivo and in vitro. Here we study the onset and maturation of Nogo-A and Nogo receptor in the entorhino-hippocampal formation of developing and adult mice. We also provide evidence that Nogo-A does not inhibit embryonic hippocampal neurons, in contrast to other cell types such as cerebellar granule cells. Our results also show that Nogo and Nogo receptor mRNA are expressed in the adult by both principal and local-circuit hippocampal neurons, and that after lesion, Nogo-A is also transiently expressed by a subset of reactive astrocytes. Furthermore, we analyzed their regulation after kainic acid (KA) treatment and in response to the transection of the entorhino-hippocampal connection. We found that Nogo-A and Nogo receptor are differentially regulated after kainic acid or perforant pathway lesions. Lastly, we show that the regenerative potential of lesioned entorhino-hippocampal organotypic slice co-cultures is increased after blockage of Nogo-A with two IN-1 blocking antibodies. In conclusion, our results show that Nogo and its receptor might play key roles during development of hippocampal connections and that they are implicated in neuronal plasticity in the adult.

Nogo receptor mRNA expression in intact and regenerating CNS neurons.

The expression of mRNA for Nogo-66 receptor (NgR) in unoperated adult rats and mice, and rats with nerve grafts placed in the thalamus and cerebellum to stimulate axonal regeneration, was investigated by in situ hybridization. NgR was strongly expressed in neurons of the neocortex, hippocampal formation, and amygdaloid nuclei and dorsal thalamus and moderately expressed in the red nucleus and vestibular nuclei. NgR mRNA was expressed in cerebellar deep nuclei and more strongly by granule cells than by Purkinje cells. Large regions of the forebrain, including the striatum, thalamic reticular nucleus, hypothalamus, and basal forebrain showed little or no NgR expression. NgR was weakly expressed in spinal neurons and some primary sensory neurons. Nerve implantation into the brain did not affect NgR expression. Some regeneration-competent neurons expressed NgR but others did not. Thus NgR expression was not correlated with the ability of neurons to regenerate axons into nerve grafts although Nogo-66 was strongly upregulated by some cells in the distal stumps of injured sciatic nerves. Nogo-66 transcripts were strongly expressed by many classes of CNS neurons and less strongly in white matter.

Nogo-A expression in the intact and injured nervous system

The expression of Nogo-A mRNA and protein in the nervous system of adult rats and cultured neurons was studied by in situ hybridisation and immunohistochemistry. Nogo-A mRNA was expressed by many cells in unoperated animals, including spinal motor, DRG, and sympathetic neurons, retinal ganglion cells, and neocortical, hippocampal, and Purkinje neurons. Nogo-A protein was strongly expressed by presumptive oligodendrocytes, but not by NG2+glia and was abundant in motor, DRG, and sympathetic neurons, retinal ganglion cells, and many Purkinje cells, but was difficult to detect in dentate gyrus neurons and some neocortical neurons. Cultured fetal mouse neocortical neurons and adult rat DRG neurons strongly expressed Nogo-A in their perikarya, growth cones, and axonal varicosities. All axons in the intact sciatic nerve contained Nogo-A and many but not all regenerating axons were strongly Nogo-A immunopositive after sciatic nerve transection. Ectopic muscle fibres that developed among the regenerating axons were also Nogo-A immunopositive. Following injury to the spinal cord, Nogo-A mRNA was upregulated around the lesion and Nogo-A protein was strongly expressed in injured dorsal column fibres and their sprouts which entered the lesion site. Following optic nerve crush, Nogo-A accumulated in the proximal and distal stumps bordering the lesions.

Chapter 17 Cellular and molecular correlates of the regeneration of adult mammalian CNS axons into peripheral nerve grafts

Studies of the regeneration of CNS axons into peripheral nerve grafts have provided information crucial to our understanding of the regenerative potential of CNS neurons. Injured axons in the thalamus and corpus striatum produce regenerative sprouts within a few days of graft implantation, apparently in response to living cells in the grafts. The regenerating axons often grow directly towards the grafts, and enter Schwann cell columns where they elongate surrounded by Schwann cell processes. The regenerating CNS axons, and the Schwann cell processes along which they grow, initially express the cell adhesion molecules NCAM, and L1. The axons also express polysialic acid and, unlike regenerating peripheral axons, bind tenascin-C derived from Schwann cells. Wherever peripheral nerve grafts are implanted into the CNS they appear to promote the differential regeneration of CNS axons. Most of the axons which grow into grafts in the thalamus are derived from the thalamic reticular nucleus (TRN), whereas grafts in the striatum promote regeneration of axons from the substantia nigra pars compacta (SNpc) and grafts in the cerebellum promote regeneration from deep cerebellar nuclei (DCN) and brainstem precerebellar neurons. In contrast most thalamocortical projection neurons, striatal projection neurons and Purkinje cells in the cerebellar cortex are poor at regenerating. There are patterns to the expression of regeneration-related molecules by axons injured by nerve grafts in the CNS. Most neurons which regenerate well (e.g. TRN and DCN neurons) upregulate GAP-43, L1 and the transcription factor c-jun in response to a graft, whereas those neurons which do not regenerate well (e.g. Purkinje cells, thalamocortical and striatal projection neurons) do not upregulate these molecules. These observations suggest that some classes of CNS neurons may be intrinsically unable to regenerate axons and the repair of injuries in the brain and spinal cord may consequently require some form of gene therapy for axotomised neurons.