ABSTRACT

A prospective first-in-human Phase 1 CRISPR gene editing trial in the United States for pa-tients with melanoma, synovial sarcoma, and multiple myeloma offers hope that gene editing tools may usefully treat human disease. An overarching ethical challenge with first-in-human Phase 1 clinical trials, however, is knowing when it is ethically acceptable to initiate such trials on the basis of safety and effi-cacy data obtained from pre-clinical studies. If the pre-clinical studies that inform trial design are them-selves poorly designed – as a result of which the quality of pre-clinical evidence is deficient – then the ethical requirement of scientific validity for clinical research may not be satisfied. In turn, this could mean that the Phase 1 clinical trial will be unsafe and that trial participants will be exposed to risk for no potential benefit. To assist sponsors, researchers, clinical investigators and reviewers in deciding when it is ethically acceptable to initiate first-in-human Phase 1 CRISPR gene editing clinical trials, structured processes have been developed to assess and minimize translational distance between pre-clinical and clinical research. These processes draw attention to various features of internal validity, construct validi-ty, and external validity. As well, the credibility of supporting evidence is to be critically assessed with particular attention to optimism bias, financial conflicts of interest and publication bias. We critically ex-amine the pre-clinical evidence used to justify the first-in-human Phase 1 CRISPR gene editing cancer trial in the United States using these tools.We conclude that the proposed trial cannot satisfy the ethical requirement of scientific validity because the supporting pre-clinical evidence used to inform trial design is deficient.

Stem cells are likely to be used as an alternate source of biological material for neural transplantation to treat Parkinson's disease in the not too distant future. Among the several ethical criteria that must be fulfilled before proceeding with clinical research, a favourable benefit to risk ratio must be obtained. The potential benefits to the participant and to society are evaluated relative to the risks in an attempt to offer the participants a reasonable choice. Through examination of preclinical studies transplanting stem cells in animals and the transplantation of fetal tissue in patients with Parkinson's disease, a current set of potential benefits and risks for neural transplantation of stem cells in clinical research of Parkinson's disease are derived. The potential benefits to research participants undergoing stem cell transplantation are relief of parkinsonian symptoms and decreasing doses of parkinsonian drugs. Transplantation of stem cells as a treatment for Parkinson's disease may benefit society by providing knowledge that can be used to help determine better treatments in the future. The risks to research participants undergoing stem cell transplantation include tumour formation, inappropriate stem cell migration, immune rejection of transplanted stem cells, haemorrhage during neurosurgery and postoperative infection. Although some of these risks are general to neurosurgical transplantation and may not be reduced for participants, the potential risk of tumour formation and inappropriate stem cell migration must be minimised before obtaining a favourable potential benefit to risk calculus and to provide participants with a reasonable choice before they enroll in clinical studies.

Low dopaminergic cell survival and suboptimal fiber reinnervation are likely major contributing factors for the limited benefits of neural transplantation in Parkinson's disease (PD) patients. Glial cell lined-derived neurotrophic factor (GDNF) has been shown to enhance dopaminergic cell survival and fiber outgrowth of the graft site as well as promote behavioral recovery in rodent models of PD, while erythropoietin (EPO) can produce dopaminergic neuroprotective effects against 6-hydroxydopamine (6-OHDA) exposure on cultured neurons and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice. The aim of this study was to determine if fetal ventral mesencephalic (FVM) tissue exposed to hibernation media containing a combination of GDNF and EPO could enhance dopaminergic graft survival, striatal reinnervation and functional recovery in a 6-OHDA rodent model of PD. FVM tissue was dissected from 14-day-old rat fetuses and placed for 6 days in hibernation media alone, and in hibernation media that received either a daily administration of GDNF, EPO or a combination of GDNF and EPO. Following hibernation, FVM cells were transplanted as a single cell suspension into the striatum of unilateral 6-OHDA-lesioned rats. Rotational behavioral assessment revealed animals that received FVM tissue exposed to GDNF, EPO or the combination of both drugs had accelerated functional recovery. Immunohistochemical and stereological assessment revealed a significant increase in graft fiber density and angiogenesis into the graft when compared with control. These findings suggest that the hibernation of FVM tissue in a combination of GDNF and EPO can enhance graft efficacy and may have important implications for tissue preparation protocols for clinical neural transplantation in PD.

Huntington's disease (HD) is a neurodegenerative disorder that is characterized by progressive dementia, choreiform involuntary movements, and emotional deterioration. Neuropathological features include the progressive degeneration of striatal γ-aminobutyric acid (GABA) neurons. New therapeutic approaches, such as the transplantation of human neural precursor cells (hNPCs) to replace damaged or degenerated cells, are currently being investigated. The aim of this study was to investigate the potential for utilizing telencephalic hNPCs expanded in suspension bioreactors for cell restorative therapy in a rodent model of HD. hNPCs were expanded in a hydrodynamically controlled and homogeneous environment under serum-free conditions. In vitro analysis revealed that the bioreactor-expanded telencephalic (BET)-hNPCs could be differentiated into a highly enriched population of GABAergic neurons. Behavioral assessments of unilateral striatal quinolinic acid-lesioned rodents revealed a significant improvement in motor and memory deficits following transplantation with GABAergic cells differentiated from BET-hNPCs. Immunohistochemical analysis revealed that transplanted BET-hNPCs retained a GABAergic neuronal phenotype without aberrant transdifferentiation or tumor formation, indicating that BET-hNPCs are a safe source of cells for transplantation. This preclinical study has important implications as the transplantation of GABAergic cells derived from predifferentiated BET-hNPCs may be a safe and feasible cell replacement strategy to promote behavioral recovery in HD.

Object Fetal tissue transplantation for Parkinson disease (PD) has demonstrated promising results in experimental and clinical studies. However, the widespread clinical application of this therapeutic approach is limited by a lack of fetal tissue. Human neural precursor cells (HNPCs) are attractive candidates for transplantation because of their long-term proliferation activity. Furthermore, these cells can be reproducibly expanded in a standardized fashion in suspension bioreactors. In this study the authors sought to determine whether the survival, differentiation, and migration of HNPCs after transplantation depended on the region of precursor cell origin, intracerebral site of transplantation, and duration of their expansion. Methods Human neural precursor cells were isolated from the telencephalon, brainstem, ventral mesencephalon, and spinal cord of human fetuses 8–10 weeks of gestational age, and their differentiation potential characterized in vitro. After expansion in suspension bioreactors, the HNPCs were transplanted into the striatum and substantia nigra of parkinsonian rats. Histological analyses were performed 7 weeks posttransplantation. Results The HNPCs isolated from various regions of the neuraxis demonstrated diverse propensities to differentiate into astrocytes and neurons and could all successfully expand under standardized conditions in suspension bioreactors. At 7 weeks posttransplantation, survival and migration were significantly greater for HNPCs obtained from the more rostral brain regions. The HNPCs differentiated predominantly into astrocytes after transplantation into the striatum or substantia nigra regions, and thus no behavioral improvement was observed. Conclusions Understanding the regional differences in HNPC properties is prerequisite to their application for PD cell restoration strategies.

The current transplantation paradigm for Parkinson's disease that places foetal dopaminergic cells in the striatum neither normalizes neuronal activity in basal ganglia structures such as the substantia nigra (SN) and subthalamic nucleus (STN) nor leads to complete functional recovery. It was hypothesized that restoration of parkinsonian deficits requires inhibition of the pathological overactivity of the STN and SN in addition to restoration of dopaminergic activity in the striatum. To achieve inhibition, a multitargeted basal ganglia transplantation strategy using GABAergic cells derived from either foetal striatal primordia (FSP) cells or human neural precursor cells (hNPCs) expanded in suspension bioreactors was investigated. In hemiparkinsonian rats, transplantation of foetal rat dopaminergic cells in the striatum in conjunction with GABAergic grafts in the STN and/or SN promoted significant improvement in forelimb akinesia and motor function compared to transplantation of intrastriatal dopaminergic grafts alone or in conjunction with undifferentiated hNPCs. In culture, FSP cells exhibited neuronal electrophysiological properties. However, recordings from GABAergic hNPCs revealed limited ionic conductances and an inability to fire action potentials. Despite this, they were almost as efficacious as FSP cells in inducing functional recovery following transplantation, suggesting that such recovery may have been mediated by secretion of GABA rather than by functional integration into the host. Thus, restoration of dopaminergic activity to the striatum in concert with inhibition of the STN and SN by GABAergic grafts may be beneficial for improving clinical outcomes in patients with Parkinson's disease and potential clinical application of this strategy may be enhanced by the use of differentiated hNPCs.

The development of new cell replacement strategies using neural stem cells (NSC) may provide an alternative and unlimited cell source for clinical neural transplantation in neurodegenerative diseases such as Parkinson's and Huntington's disease. The clinical application of neural transplantation using NSC will therefore depend upon the availability of clinical grade NSC that are generated in unlimited quantities in a standardized manner. In order to investigate the utility of NSC in clinical neural transplantation, undifferentiated murine NSC were first expanded for an extended period of time in suspension bioreactors containing a serum-free medium. Following expansion in suspension bioreactors, NSC were still able to differentiate in vitro into both astrocytes and neurons after exposure to brain-derived neurotrophic factor (BDNF), suggesting that bioreactor expansion does not alter cell lineage potentiality. Undifferentiated bioreactor-expanded NSC were then transplanted into the rodent striatum. Immunohistochemical examination revealed undifferentiated bioreactor-expanded NSC survived transplantation for up to 8 weeks and expressed the astrocytic immunohistochemical marker glial fibrillary acidic protein (GFAP), suggesting that the host striatal environment influences NSC cell fate upon transplantation. Moreover, no tumor formation was observed within the graft site, indicating that NSC expanded in suspension bioreactors for an extended period of time are a safe source of tissue for transplantation. Future studies should focus on predifferentiating NSC towards specific neuronal phenotypes prior to transplantation in order to restore behavioral function in rodent models of neurodegenerative disease.