Compliance in Non-Clinical Development of Cell-, Gene-, and Tissue-Based Medicines: Good Practice for Better Therapies

Non-clinical safety; Product development; Quality complianceSeguridad no clínica; Desarrollo de productos; Cumplimiento de calidadSeguretat no clínica; Desenvolupament de productes; Compliment de la qualitatThe development of cell-, gene- and tissue engineering (CGT)-based therapies must adhere to strict pharmaceutical quality management standards, as for any other biological or small-molecule drug. However, early developments often failed to fully comply with good laboratory practices (GLP) in non-clinical safety studies. Despite an upward trend of positive opinions in marketing authorization applications, evidence of adherence to the principles of GLP is not openly reported; therefore, their relative impact on the overall quality of the product development program is unknown. Herein we investigated the actual degree of GLP implementation and the underlying factors impeding full compliance in non-clinical developments of CGT-based marketed medicines in the EU and USA, including (i) the co-existence of diverse quality management systems of more strategic value for small organizations, particularly current Good Manufacturing Practices n(GMP); (ii) lack of regulatory pressure to pursue GLP certification; and (iii) the involvement of public institutions lacking a pharmaceutical mindset and resources. As a final reflection, we propose conformity to good research practice criteria not as a doctrinaire impediment to scientific work, but as a facilitator of efficient clinical translation of more effective and safer innovative therapies.The authors received no specific funding for this work, which has been developed in the context of Red Española de Terapias Avanzadas (TERAV, expedient no.’s RD21/0017/0015 and RD21/0017/0022) funded by Instituto de Salud Carlos III (ISCIII) in the context of NextGenerationEU’s Recovery, Transformation and Resilience Plan

Sox-2 Positive Neural Progenitors in the Primate Striatum Undergo Dynamic Changes after Dopamine Denervation.

The existence of endogenous neural progenitors in the nigrostriatal system could represent a powerful tool for restorative therapies in Parkinson's disease. Sox-2 is a transcription factor expressed in pluripotent and adult stem cells, including neural progenitors. In the adult brain Sox-2 is expressed in the neurogenic niches. There is also widespread expression of Sox-2 in other brain regions, although the neurogenic potential outside the niches is uncertain. Here, we analyzed the presence of Sox-2+ cells in the adult primate (Macaca fascicularis) brain in naïve animals (N = 3) and in animals exposed to systemic administration of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine to render them parkinsonian (N = 8). Animals received bromodeoxyuridine (100 mg/kg once a day during five consecutive days) to label proliferating cells and their progeny. Using confocal and electron microscopy we analyzed the Sox-2+ cell population in the nigrostriatal system and investigated changes in the number, proliferation and neurogenic potential of Sox-2+ cells, in control conditions and at two time points after MPTP administration. We found Sox-2+ cells with self-renewal capacity in both the striatum and the substantia nigra. Importantly, only in the striatum Sox-2+ was expressed in some calretinin+ neurons. MPTP administration led to an increase in the proliferation of striatal Sox-2+ cells and to an acute, concomitant decrease in the percentage of Sox-2+/calretinin+ neurons, which recovered by 18 months. Given their potential capacity to differentiate into neurons and their responsiveness to dopamine neurotoxic insults, striatal Sox-2+ cells represent good candidates to harness endogenous repair mechanisms for regenerative approaches in Parkinson's disease

Fast and efficient neural conversion of human hematopoietic cells

Neurons obtained directly from human somatic cells hold great promise for disease modeling and drug screening. Available protocols rely on overexpression of transcription factors using integrative vectors and are often slow, complex, and inefficient. We report a fast and efficient approach for generating induced neural cells (iNCs) directly from human hematopoietic cells using Sendai virus. Upon SOX2 and c-MYC expression, CD133-positive cord blood cells rapidly adopt a neuroepithelial morphology and exhibit high expansion capacity. Under defined neurogenic culture conditions, they express mature neuronal markers and fire spontaneous action potentials that can be modulated with neurotransmitters. SOX2 and c-MYC are also sufficient to convert peripheral blood mononuclear cells into iNCs. However, the conversion process is less efficient and resulting iNCs have limited expansion capacity and electrophysiological activity upon differentiation. Our study demonstrates rapid and efficient generation of iNCs from hematopoietic cells while underscoring the impact of target cells on conversion efficiency

iPS cell cultures from a Gerstmann-Straussler-Scheinker patient with the Y218N PRNP mutation recapitulate tau pathology

Gerstmann-Straussler-Scheinker (GSS) syndrome is a fatal autosomal dominant neurodegenerative prionopathy clinically characterized by ataxia, spastic paraparesis, extrapyramidal signs and dementia. In some GSS familiar cases carrying point mutations in the PRNP gene, patients also showed comorbid tauopathy leading to mixed pathologies. In this study we developed an induced pluripotent stem (iPS) cell model derived from fibroblasts of a GSS patient harboring the Y218N PRNP mutation, as well as an age-matched healthy control. This particular PRNP mutation is unique with very few described cases. One of the cases presented neurofibrillary degeneration with relevant Tau hyperphosphorylation. Y218N iPS-derived cultures showed relevant astrogliosis, increased phospho-Tau, altered microtubule-associated transport and cell death. However, they failed to generate proteinase K-resistant prion. In this study we set out to test, for the first time, whether iPS cell-derived neurons could be used to investigate the appearance of disease-related phenotypes (i.e, tauopathy) identified in the GSS patient

Estudio de la lesion nigroestriatal inducida por la inyeccion de 1-metil-4-fenilpiridina en estriado en la rata Un modelo para investigacion en la enfermedad de parkinson

Centro de Informacion y Documentacion Cientifica (CINDOC). C/Joaquin Costa, 22. 28002 Madrid. SPAIN / CINDOC - Centro de Informaciòn y Documentaciòn CientìficaSIGLEESSpai

Cellular Programming and Reprogramming: Sculpting Cell Fate for the Production of Dopamine Neurons for Cell Therapy

Pluripotent stem cells are regarded as a promising cell source to obtain human dopamine neurons in sufficient amounts and purity for cell replacement therapy. Importantly, the success of clinical applications depends on our ability to steer pluripotent stem cells towards the right neuronal identity. In Parkinson disease, the loss of dopamine neurons is more pronounced in the ventrolateral population that projects to the sensorimotor striatum. Because synapses are highly specific, only neurons with this precise identity will contribute, upon transplantation, to the synaptic reconstruction of the dorsal striatum. Thus, understanding the developmental cell program of the mesostriatal dopamine neurons is critical for the identification of the extrinsic signals and cell-intrinsic factors that instruct and, ultimately, determine cell identity. Here, we review how extrinsic signals and transcription factors act together during development to shape midbrain cell fates. Further, we discuss how these same factors can be applied in vitro to induce, select, and reprogram cells to the mesostriatal dopamine fate

Human Neural Stem Cells for Cell-Based Medicinal Products

Neural stem cells represent an attractive tool for the development of regenerative therapies and are being tested in clinical trials for several neurological disorders. Human neural stem cells can be isolated from the central nervous system or can be derived in vitro from pluripotent stem cells. Embryonic sources are ethically controversial and other sources are less well characterized and/or inefficient. Recently, isolation of NSC from the cerebrospinal fluid of patients with spina bifida and with intracerebroventricular hemorrhage has been reported. Direct reprogramming may become another alternative if genetic and phenotypic stability of the reprogrammed cells is ensured. Here, we discuss the advantages and disadvantages of available sources of neural stem cells for the production of cell-based therapies for clinical applications. We review available safety and efficacy clinical data and discuss scalability and quality control considerations for manufacturing clinical grade cell products for successful clinical application