Adeno-associated viral vectors for clinical gene transfer studies

Recombinant adeno-associated viral (rAAV) vectors can mediate the safe and long-term correction of genetic diseases in animal models following a single administration. These pre-clinical studies are the basis of human trials that have shown rAAV vector persistence and safety in humans following delivery to lung, sinus, skeletal muscle, brain and liver. Transient disease correction has also been demonstrated in humans treated for hemophilia B and cystic fibrosis using AAV2 vectors. The physiochemical properties of rAAV vector virions are amenable to industry accepted manufacturing methodologies, long-term storage and direct in vivo administration. Recombinant adeno-associated virus vectors are manufactured in compliance with current Good Manufacturing Practices (cGMPs) as outlined in the Code of Federal Regulations (21CFR). To meet these requirements, manufacturing controls and quality systems are established, including 1) adequate facilities and equipment, 2) personnel who have relevant education or experience and are trained for specific assigned duties, 3) raw materials that are qualified for use and 4) a process (including production, purification, formulation, filling, storage and shipping) that is controlled, aseptic, reliable and consistent. Quality systems including Quality Control (QC) and Quality Assurance (QA) are also implemented. These manufacturing procedures and quality systems are designed so the product meets its release specifications to ensure that patients receive a safe, pure, potent and stable investigational drug.     

Astrocytes in injured adult rat spinal cord may acquire the potential of neural stem cells

It has been well documented that in adult rats astrocytes in the subventricular zone and subgranular layer of the dentate gyrus are neural stem cells. Elsewhere in the CNS astrocytes are not generally recognized as stem cells. Here we describe nestin expression in a population of astrocytes in the spinal cord of adult rat following cord injury. In either hemitransectioned or longitudinally cut spinal cord, there was widespread nestin expression in astrocytes of both the gray and white matters. Isolation of the lateral part of the spinal cord from the central canal region, where stem cells may reside, could not block the appearance of nestin-immunoreactive astrocytes in the lateral cord, and none of them showed Fast DiI labeling after the central canal ependyma had been labeled by the dye, indicating that the nestin-immunoreactive astrocytes can evolve locally in the lateral cord. They were found to be undergoing a process of de-differentiation. Culture of the nestin-immunoreactive astrocytes of the lateral cord generated neurospheres, the cells of which had the ability of self-renewal, and were able to differentiate into neurons, astrocytes, or oligodendrocytes. Taken together, the results indicate that the astrocytes in injured adult rat spinal cord may acquire the potential of neural stem cells.     

Site-specific gene transfer into the rat spinal cord by photomechanical waves

Nonviral, site-specific gene delivery to deep tissue is required for gene therapy of a spinal cord injury. However, an efficient method satisfying these requirements has not been established. This study demonstrates efficient and targeted gene transfer into the spinal cord by using photomechanical waves (PMWs), which were generated by irradiating a black laser absorbing rubber with 532-nm nanosecond Nd:YAG laser pulses. After a solution of plasmid DNA coding for enhanced green fluorescent protein (EGFP) or luciferase was intraparenchymally injected into the spinal cord, PMWs were applied to the target site. In the PMW application group, we observed significant EGFP gene expression in the white matter and remarkably high luciferase activity only in the spinal cord segment exposed to the PMWs. We also assessed hind limb movements 24 h after the application of PMWs based on the Basso-Beattie-Bresnahan (BBB) score to evaluate the noninvasiveness of this method. Locomotor evaluation showed no significant decrease in BBB score under optimum laser irradiation conditions. These findings demonstrated that exogenous genes can be efficiently and site-selectively delivered into the spinal cord by applying PMWs without significant locomotive damage.     

Retraining the injured spinal cord

The present review presents a series of concepts that may be useful in developing rehabilitative strategies to enhance recovery of posture and locomotion following spinal cord injury. First, the loss of supraspinal input results in a marked change in the functional efficacy of the remaining synapses and neurons of intraspinal and peripheral afferent (dorsal root ganglion) origin. Second, following a complete transection the lumbrosacral spinal cord can recover greater levels of motor performance if it has been exposed to the afferent and intraspinal activation patterns that are associated with standing and stepping. Third, the spinal cord can more readily reacquire the ability to stand and step following spinal cord transection with repetitive exposure to standing and stepping. Fourth, robotic assistive devices can be used to guide the kinematics of the limbs and thus expose the spinal cord to the new normal activity patterns associated with a particular motor task following spinal cord injury. In addition, such robotic assistive devices can provide immediate quantification of the limb kinematics. Fifth, the behavioural and physiological effects of spinal cord transection are reflected in adaptations in most, if not all, neurotransmitter systems in the lumbosacral spinal cord. Evidence is presented that both the GABAergic and glycinergic inhibitory systems are up-regulated following complete spinal cord transection and that step training results in some aspects of these transmitter systems being down-regulated towards control levels. These concepts and observations demonstrate that (a) the spinal cord can interpret complex afferent information and generate the appropriate motor task; and (b) motor ability can be defined to a large degree by training.     

Advances in vector-mediated gene transfer

Clinical applications of gene transfer technology initially targeted the treatment of inherited monogenetic disorders and cancers refractory to conventional therapies. Today, gene transfer approaches are being developed for most tissues and for multiple disorders including those affecting quality of life. The focus herein is eventual application of gene transfer technology for the management of organ-directed autoimmunity. A specific example is presented: Sjögren’s syndrome and localized salivary gland gene transfer. The status of relevant pre-clinical gene transfer studies is reviewed, with an emphasis on use of adenoviral and adeno-associated viral vectors. Current limitations of effective organ-directed gene transfer are also discussed.     

Effectiveness of riluzole in suppressing spasticity in the spinal cord injured rat

Spasticity poses a major detrimental impact on the quality of life in a significant number of people with spinal cord injury (SCI). Recent observations in our laboratory suggest that spinal transection at the sacral S₂ level induces a significant increase in glutamatergic input to sacrocaudal motoneurons during the time spasticity is present in the tail muscles. The present study examined the effectiveness of riluzole, an agent that has been shown to reduce glutamate release, in managing spasticity within the tail musculature. In this blinded, cross-over study animals with S₂ spinal transections were tested behaviorally for the progression of spasticity in the tail musculature using our established system. When the animals demonstrated a significant level of spastic behavior (e.g. increased response to quick stretch, noxious and non-noxious cutaneous stimuli), they received either saline or riluzole (8 or 10 mg/kg i.p.) and assessed behaviorally at 1, 3, 6, and 12 post-injection. Results: riluzole at 8 mg/kg significantly decreased the response of the tail muscle to noxious and non-noxious cutaneous stimuli for the first 3 h post-administration, while administration of riluzole at 10 mg/kg significantly decreased the responsiveness of the tail to all of the behavioral assessments. However, a significant percentage of the animals displayed motor impairments at this higher dosage. Conclusion: suppression of glutamate release by the administration of riluzole can reduce several, but not all, aspects of spastic activity in the tail muscles at concentrations that do not elicit negative side-effects.     

Adeno-associated viral vectors for retinal gene transfer and treatment of retinal diseases

Retinal gene transfer holds big promises for the treatment of inherited and non-inherited blinding diseases, such as retinitis pigmentosa or age-related macular degeneration. Key to the development of successful gene-based therapies for the eye are efficient tools for retinal gene transfer. Vectors based on adeno-associated viruses (AAV) are able to transduce robustly and persistently different retinal cell types of animal models after a single intraocular administration. Recombinant AAV (rAAV) vectors are versatile gene transfer tools in that capsid proteins from dozens of AAV serotypes can be easily interchanged, resulting in the creation of recombinant vectors with unique transduction properties. This has allowed successful proof-of-principle studies using rAAV-mediated gene transfer to restore retinal morphology and function in small and large animal models of retinal diseases. In addition, gene delivery using rAAV vectors in the eye seems to have appropriate biosafety characteristics to rapidly move it from bench to bedside. All the above aspects will be reviewed and discussed in detail below.     

Therapeutic time window for methylprednisolone in spinal cord injured rat.

Recent clinical trials have reported that methylprednisolone sodium succinate administered within 8 hours improves neurological recovery in human spinal cord injury (SCI). Methylprednisolone, however, was ineffective and possibly even deleterious when given more than 8 hours after injury. This finding suggests that a therapeutic time window exists in spinal cord injury. In order to determine the doses, durations and timing of methylprednisolone treatment for optimal neuroprotection, a single or two bolus dose of methylprednisolone (30 mg/kg) was administered at 10, 30, 120, 150 and 240 min. after three graded spinal cord injury. The primary outcome measure was 24-hour spinal cord lesion volumes estimated from spinal cord Na+ and K+ shifts. A single 30 mg/kg dose of methylprednisolone at 10 min. after injury significantly reduced 24-hour lesion volumes in injured rat spinal cords. However, any other methylprednisolone treatment starting 30 min. or more after injury had no effect on 24-hour lesion volumes compared to the vehicle control group. Moreover, delayed treatment increased lesion volumes in some cases. These results suggest that the NYU SCI model has a very short therapeutic window.     

Cell division in injured spinal cord

Thymidine-H³ radioautography was used to study the proliferative response to penetrating wounds of the mouse spinal cord. In one group of animals mononuclear leukocytes which infiltrate nervous tissue wounds were labeled by injecting thymidine-H³ prior to injury. In two other experimental groups the cells which synthesized DNA in the nervous tissue following the injury were labeled by giving either a single injection of isotope shortly before sacrifice or by giving four injections during the 24 hours prior to sacrifice. The animals were sacrificed over a five day period following spinal cord injury. Although the labeled nuclei in all three groups were similar in appearance, their distribution about the lesion was very different. The labeled blood cells were greatly concentrated at the wound, while the cells that responded to injury by DNA synthesis were much more evenly spread throughout the tissue. When these distributions were converted to straight lines and compared statistically, there was a very low probability that the group of cells labeled before injury and the two groups labeled after injury were samples from the same population. Although mononuclear leukocytes do proliferate in and around nervous tissue wounds, other cells originally present in the nervous tissue wounds, other cells originally present in the nervous tissue must also proliferate.     

Exercise-induced gene expression changes in the rat spinal cord

There is growing evidence that exercise benefits recovery of neuromuscular function from spinal cord injury (SCI). However, the effect of exercise on gene expression in the spinal cord is poorly understood. We used oligonucleotide microarrays to compare thoracic and lumbar regions of spinal cord of either exercising (voluntary wheel running for 21 days) or sedentary rats. The expression data were filtered using statistical tests for significance, and K-means clustering was then used to segregate lists of significantly changed genes into sets based upon expression patterns across all experimental groups. Levels of brain-derived neurotrophic factor (BDNF) protein were also measured after voluntary exercise, across different regions of the spinal cord. BDNF mRNA increased with voluntary exercise, as has been previously shown for other forms of exercise, contributed to by increases in both exon I and exon III. The exercise-induced gene expression changes identified by microarray analysis are consistent with increases in pathways promoting neuronal health, signaling, remodeling, cellular transport, and development of oligodendrocytes. Taken together these data suggest cellular pathways through which exercise may promote recovery in the SCI population.     

The injured spinal cord spontaneously forms a new intraspinal circuit in adult rats

In contrast to peripheral nerves, central axons do not regenerate. Partial injuries to the spinal cord, however, are followed by functional recovery. We investigated the anatomical basis of this recovery and found that after incomplete spinal cord injury in rats, transected hindlimb corticospinal tract (CST) axons sprouted into the cervical gray matter to contact short and long propriospinal neurons (PSNs). Over 12 weeks, contacts with long PSNs that bridged the lesion were maintained, whereas contacts with short PSNs that did not bridge the lesion were lost. In turn, long PSNs arborize on lumbar motor neurons, creating a new intraspinal circuit relaying cortical input to its original spinal targets. We confirmed the functionality of this circuit by electrophysiological and behavioral testing before and after CST re-lesion. Retrograde transynaptic tracing confirmed its integrity, and revealed changes of cortical representation. Hence, after incomplete spinal cord injury, spontaneous extensive remodeling occurs, based on axonal sprout formation and removal. Such remodeling may be crucial for rehabilitation in humans.     

人发角蛋白植入对大鼠脊髓损伤功能与形态的影响

目的：研究脊髓损伤后植入人发角蛋白（human hair keratin，HHK）对动物行为学影响及其对损伤脊髓的形态学影响与HHK本身的形态学变化。方法：采用重物下落撞击法制备大鼠脊髓损伤模型，在损伤处移植入HHK，分别于植入后5、10、15、20、25、30d进行行为学检查，于20d与30d脊髓进行形态学观察。结果：HHK植入后在30d内对大鼠的行为学影响与模型对照组没有统计学意义，但HHK可降解吸收融入损伤的脊髓组织，而受损伤的脊髓组织中可见大量胶质细胞、成纤维细胞与巨噬细胞浸润、神经纤维与髓鞘朗革非氏细胞。结论：HHK可能在脊髓损伤后的修复与重建中起作用。     

Retroviral vector-mediated gene transfer into umbilical cord blood-derived megakaryocyte and platelet progenitors.

Optimizing platelet engraftment following hematopoietic stem cell transplantation is essential for minimizing transplant-related morbidity, particularly following umbilical cord blood transplantation (UCBT), where platelet engraftment frequently takes >60 days. One strategy for optimizing platelet engraftment following UCBT is to study or alter the genetic program of megakaryocyte/platelet (Mk/plt) progenitors. Retroviral vector gene transfer has previously proven useful for studying the biology of hematopoietic stem cells; however, procedures for transducing UCB cells of the Mk/plt lineage with retroviral vectors have not been described. We report here that Mk/plt progenitors generated from UCB progenitors can be efficiently transduced with retroviral vectors. Transduced Mk/plt cells were identified and quantitated by expression of a vector transgene encoding a truncated version of the human nerve growth factor receptor (NGFR). Vector-mediated NGFR expression could be readily detected in Mk/plt progenitors defined by immunophenotypic, morphologic, and functional criteria. In addition, NGFR expression persisted in mature anucleate platelets generated from the transduced Mk/plt progenitors. These methods may be useful for introducing genetic elements into Mk/plt progenitors to study various aspects of platelet development and biology and for marking ex vivo expanded Mk/plt progenitors to determine their contribution to engraftment.     

Imaging corticospinal tract connectivity in injured rat spinal cord using manganese-enhanced MRI

Background  

Manganese-enhanced MRI (MEI) offers a novel neuroimaging modality to trace corticospinal tract (CST) in live animals. This paper expands this capability further and tests the utility of MEI to image axonal fiber connectivity in CST of injured spinal cord (SC).     

Peripheral neuropathy in spinal cord injured patients

We performed electrophysiological studies on the lower limbs of a group of clinically complete spinal cord injured patients. Our findings indicated a frequent asymmetric sensory and motor nerve involvement at proximal and distal sites. We suggest that mechanical compression and traction is one of etiological factors for such findings.     

微囊化兔雪旺细胞移植对大鼠损伤脊髓胶质纤维酸性蛋白表达的影响

目的:观察大鼠脊髓损伤后及进行微囊化兔雪旺细胞移植后胶质纤维酸性蛋白(GFAP)表达的变化.方法: 130只成年SD大鼠随机分为微囊组、细胞悬液组、损伤对照组和正常对照组4组,术后3、 7、 14及28d,冰冻切片行免疫组织化学显色观察GFAP表达的变化.结果:大鼠脊髓损伤后3～14d, GFAP阳性细胞数及平均光密度均增加;至第28天时则减少,但仍高于正常组.其中阳性细胞数和平均光密度在第7天开始,微囊组与细胞悬液组、损伤组比较均有明显降低.结论: 微囊化异种雪旺细胞移植能抑制损伤脊髓GFAP的表达,减轻由反应性胶质化所形成的胶质瘢痕.     

Glutamate-activated channels in adult rat ventral spinal cord cells.

1. Currents in response to rapid application of glutamate and its agonists were studied in cells dissociated from the ventral spinal cord of adult rats. 2. Glutamate activated an inward current that desensitized in less than 15 ms. 3. Responses to quisqualate and to DL-alpha-amino-3-hydroxy-5-methyl-isoxeazolepropionic acid (AMPA) also desensitized with time constants ranging from 7 to 18 ms in whole cell configuration and from 3.4 to 4.3 ms in outside-out configuration. Desensitization rate was independent of membrane potential. Single-channel conductance was 12 pS. 4. Currents in response to N-methyl-D-aspartate activation also desensitized; the time constants ranged from 15 to 50 ms. Single-channel conductance was 23 pS. 5. Kainate responses did not desensitize appreciably. Single-channel conductance was 17 pS. 6. These data obtained from adult cells are similar to values reported for cultured embryonic and neonatal neurons, indicating minimal postnatal changes in these aspects of glutamate receptors.