Regulatory systems and requirements for clinical trials of AAV-based gene therapies - Perspectives from six Asian countries or regions: Report from the 6th Asia Partnership Conference of Regenerative Medicine - April 20, 2023.

Gene therapies, which include viral-vector gene delivery, genome editing, and genetically modified cell therapy, are innovative treatments with the potential to address the underlying genetic causes of disorders and to provide life-changing value in terms of curing disease. Although adeno-associated virus (AAV)-based gene therapy is one of the most advanced types of gene therapy, far fewer AAV-based gene therapy studies have been conducted in Asia than in North America and Europe. The 6th Asia Partnership Conference of Regenerative Medicine (APACRM) was held on April 20, 2023 in Tokyo, Japan. APACRM Working Group 3 comprehensively analyzed the regulatory processes that occur prior to the initiation of clinical trials as well as the regulatory requirements for AAV-based gene therapies for six Asian countries or regions (China, India, Japan, Singapore, South Korea, and Taiwan). In this article, we report the outcomes of this conference, summarizing the regulatory requirements for initiating clinical trials for AAV-based gene therapies in terms of the laws, regulations, and guidelines for gene therapy; consultations or reviews required by the health authorities; points to consider for scientific reviews by the health authorities; and specific challenges to address when developing gene therapy products in these locations. Finally, we present several policy recommendations, including simplifying the regulatory review system for multiple scientific review areas; simplifying the regulatory consultation system; and providing training programs and regulatory guidance to support the advancement of gene therapy development in Asia.

The Application of an Omentum Graft or Flap in Spinal Cord Injury.

BACKGROUND: Spinal cord injury (SCI) causes a primary injury at the lesion site and triggers a secondary injury and prolonged inflammation. There has been no definitive treatment till now. Promoting angiogenesis is one of the most important strategies for functional recovery after SCI. The omentum, abundant in blood and lymph vessels, possesses the potent ability of tissue regeneration. METHODS: The present work examines the efficacy of autologous omentum, either as a flap (with vascular connection intact) or graft (severed vascular connection), on spinal nerve regeneration. After contusive SCI in rats, a thin sheath of omentum was grafted to the injured spinal cord. RESULTS: Omental graft improved behavior scores significantly from the 3rd to 6th week after injury (6th week, 5.5 ± 0.5 vs. 8.6 ± 1.3, p < 0.05). Furthermore, the reduction in cavity and the preservation of class III ß-tubulin-positive nerve fibers in the injury area was noted. Next, the free omental flap was transposed to a completely transected SCI in rats through a pre-implanted tunnel. The flap remained vascularized and survived well several weeks after the operation. At 16 weeks post-treatment, SCI rats with omentum flap treatment displayed the preservation of significantly more nerve fibers (p < 0.05) and a reduced injured cavity, though locomotor scores were similar. CONCLUSIONS: Taken together, the findings of this study indicate that treatment with an omental graft or transposition of an omental flap on an injured spinal cord has a positive effect on nerve protection and tissue preservation in SCI rats. The current data highlight the importance of omentum in clinical applications.

Local inhibition of matrix metalloproteinases reduced M2 macrophage activity and impeded recovery in spinal cord transected rats after treatment with fibroblast growth factor-1 and nerve grafts.

Alternatively activated macrophages (M2 macrophages) promote central nervous system regeneration. Our previous study demonstrated that treatment with peripheral nerve grafts and fibroblast growth factor-1 recruited more M2 macrophages and improved partial functional recovery in spinal cord transected rats. The migration of macrophages is matrix metalloproteinase (MMP) dependent. We used a general inhibitor of MMPs to influence macrophage migration, and we examined the migration of macrophage populations and changes in spinal function. Rat spinal cords were completely transected at T8, and 5 mm of spinal cord was removed (group T). In group R, spinal cord-transected rats received treatment with fibroblast growth factor-1 and peripheral nerve grafts. In group RG, rats received the same treatment as group R with the addition of 200 µM GM6001 (an MMP inhibitor) to the fibrin mix. We found that MMP-9, but not MMP-2, was upregulated in the graft area of rats in group R. Local application of the MMP inhibitor resulted in a reduction in the ratio of arginase-1 (M2 macrophage subset)/inducible nitric oxide synthase-postive cells. When the MMP inhibitor was applied at 8 weeks postoperation, the partial functional recovery observed in group R was lost. This effect was accompanied by a decrease in brain-derived neurotrophic factor levels in the nerve graft. These results suggested that the arginase-1 positive population in spinal cord transected rats is a migratory cell population rather than the phenotypic conversion of early iNOS+ cells and that the migration of the arginase-1+ population could be regulated locally. Simultaneous application of MMP inhibitors or promotion of MMP activity for spinal cord injury needs to be considered if the coadministered treatment involves M2 recruitment.

Contusion Spinal Cord Injury Rat Model.

Spinal cord injury (SCI) can lead to severe disability, paralysis, neurological deficits and even death. In humans, most spinal cord injuries are caused by transient compression or contusion of the spinal cord associated with motor vehicle accidents. Animal models of contusion mimic the typical SCI's found in humans and these models are key to the discovery of progressive secondary tissue damage, demyelination, and apoptosis as well as pathophysiological mechanisms post SCI. Here we describe a method for the establishment of an efficient and reproducible contusion model of SCI in adult rat.

The immunomodulator decoy receptor 3 improves locomotor functional recovery after spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) causes loss of neurons and axons and results in motor and sensory function impairments. SCI elicits an inflammatory response and induces the infiltration of immune cells, predominantly macrophages, to the injured site. Decoy receptor 3 (DcR3), also known as tumor necrosis factor receptor superfamily member (TNFRSF)-6B, is a pleiotropic immunomodulator capable of inducing macrophage differentiation into the M2 phenotype and enhancing angiogenesis. Because M2 macrophages are crucial for the recovery of impaired motor functions, we ask whether DcR3 is beneficial for the functional recovery of locomotion in Sprague-Dawley (SD) rats after SCI. METHODS: Contusion injury of the spinal cord was performed using a New York University impactor at the ninth thoracic vertebrae, followed by intrathecal injection of 15 µg recombinant protein comprising DcR3 (DcR3.Fc) in 5 µl of normal saline as the treatment, or 5 µl of normal saline as the control, into the injury epicenter. Functional recovery was evaluated using an open-field test weekly up to 6 weeks after injury. The cavity size and myelin sparing in the rostral-to-caudal region, including the epicenter of the injury, were then examined in SCI rats by histological staining. The expression of anti-inflammatory cytokines and the presence of M2 macrophages were determined by quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry at 7 day after SCI. Statistical analysis was performed using a two-tailed Student's t test. RESULTS: Intrathecal administration of DcR3.Fc significantly improved locomotor function and reduced secondary injury with a smaller wound cavity and increased myelin sparing at the lesion site. Compared with the control group, DcR3.Fc-treated rats had increased vascularization at the injury epicenter along with higher levels of interleukin (IL)-4 and IL-10 and lower level of IL-1ß on DcR3.Fc-treated rats at day 7 after SCI. Moreover, higher levels of arginase I (Arg I) and CD206 (M2 macrophage markers) and RECA-1 (endothelial marker) were observed in the epicenter on day 7 after SCI by immunofluorescence staining. CONCLUSIONS: These results indicated that DcR3.Fc may promote the M2 macrophage infiltration and enhanced angiogenesis at the lesion site, thus preserving a greater amount of spinal cord tissues and enhancing functional recovery after SCI.

Adeno-associated virus-mediated human acidic fibroblast growth factor expression promotes functional recovery of spinal cord-contused rats.

BACKGROUND: Following spinal cord injury, the delivery of neurotrophic factors to the injured spinal cord has been shown to promote axonal regeneration and functional recovery. In previous studies, we showed that acidic fibroblast growth factor (aFGF) is a potent neurotrophic factor that promotes the regeneration of axotomized spinal cord or dorsal root ganglion neurones. METHODS: We constructed a recombinant adeno-associated virus (AAV) vector to express human aFGF and evaluated aFGF expression and function in AAV-aFGF-infected PC12 cells. We analyzed AAV-green fluorescent protein (GFP) tropism and AAV-mediated aFGF expression in contused spinal cords. Animals received behavioural testing to evaluate the functional recovery. RESULTS: Overexpression of aFGF was shown in AAV-aFGF-infected PC12 cells in a dose-dependent manner. Concurrently, neurite extension and cell number were significantly increased in AAV-aFGF infected cells. AAV-mediated GFP expression persisted for at least 5 weeks in contused spinal cords, and the most prominently transduced cells were neurones. Contusive injury reduced endogenous aFGF expression in spinal cords. Overexpression of aFGF was demonstrated in AAV-aFGF transduced spinal cords compared to AAV-GFP transduced spinal cords at 3 and 14 days post-injury. Evaluation of motor function revealed that the improvement of AAV-aFGF-treated rats was prominent. Both AAV-aFGF- and recombinant human aFGF-treated rats revealed significantly better recovery at 5 weeks post-injury, compared to vehicle- and AAV-GFP-treated rats. CONCLUSIONS: These data suggest that supplement of aFGF improve the functional recovery of spinal cord-contused rats and that AAV-aFGF-mediated gene transfer could be a clinically feasible therapeutic approach for patients after nervous system injuries.

Acid fibroblast growth factor and peripheral nerve grafts regulate Th2 cytokine expression, macrophage activation, polyamine synthesis, and neurotrophin expression in transected rat spinal cords.

Spinal cord injury elicits an inflammatory response that recruits macrophages to the injured spinal cord. Quantitative real-time PCR results have shown that a repair strategy combining peripheral nerve grafts with acidic fibroblast growth factor (aFGF) induced higher interleukin-4 (IL-4), IL-10, and IL-13 levels in the graft areas of rat spinal cords compared with transected spinal cords at 10 and 14 d. This led to higher arginase I-positive alternatively activated macrophage (M2 macrophage) responses. The gene expression of several enzymes involved in polyamine biosynthesis pathways was also upregulated in the graft areas of repaired spinal cords. The treatment induced a twofold upregulation of polyamine levels at 14 d, as confirmed by HPLC. Polyamines are important for the repair process, as demonstrated by the observation that treatment with inhibitors of arginase I and ornithine decarboxylase attenuates the functional recoveries of repaired rats. After 14 d, the treatment also induced the expression of neurotrophin nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), as well as M2 macrophages within grafted nerves expressing BDNF. IL-4 was upregulated in the injury sites of transected rats that received aFGF alone compared with those that received nerve grafts alone at 10 d. Conversely, nerve graft treatment induced NGF and BDNF expression at 14 d. Macrophages expressing polyamines and BDNF may benefit axonal regeneration at 14 d. These results indicate that aFGF and nerve grafts regulate different macrophage responses, and M2 macrophages may play an important role in axonal regeneration after spinal cord injury in rats.

Dual effect of adenovirus-mediated transfer of BMP7 in mixed neuron-glial cultures: neuroprotection and cellular differentiation.

Bone morphogenetic proteins (BMPs), members of the TGF-beta superfamily, have been implicated in nervous system development and in response to injury. Previous studies have shown that recombinant BMP7 can enhance dendritic growth and protect cultured neurons from oxidative stress. Because of the presence of extracellular BMP antagonists, BMP7 seems to act locally. Therefore, the present study uses BMP7 overexpression using adenovirus (Ad)-mediated gene transfer to examine its effect in mixed neuronal cultures. Enhanced BMP7 expression selectively induces neuronal CGRP expression in a time-dependent manner. BMP7 overexpression not only significantly protects cultures from H2O2 toxicity but reduces lipopolysaccharide (LPS) stimulation. Concurrently, it profoundly reduces microglial numbers, but increases oligodendroglial and endothelial cells. Together, low-dose and continuously expressed BMP7 is both neuroprotective and differentiation-inductive.

The combination of peripheral nerve grafts and acidic fibroblast growth factor enhances arginase I and polyamine spermine expression in transected rat spinal cords.

Treatment with a combination of peripheral nerve grafts and acidic fibroblast growth factor improves hind limb locomotor function after spinal cord transection. This study examined the effect of treatment on expression of arginase I (Arg I) and polyamines. Arg I expression was low in the spinal cords of normal rats but increased following spinal injury. Only fully repaired spinal cords expressed higher Arg I levels 6-14 days following repair. In 10-day repaired spinal cords, high Arg I immunoreactivity was detected in motoneurons and alternatively activated macrophages in the graft area and graft-stump edges, and high levels of the polyamine spermine were expressed by macrophages within the intercostal nerve graft. Thus, in addition to enhancing the expression of Arg I and spermine in repaired spinal cords, our treatment may recruit activated macrophages and create a more favorable environment for axonal regrowth.