Cerebral Organoids: A Human Model for AAV Capsid Selection and Therapeutic Transgene Efficacy in the Brain

Abstract

The development of gene therapies for central nervous system disorders is challenging because it is difficult to translate preclinical data from current in vitro and in vivo models to the clinic. Therefore, we developed induced pluripotent stem cell (iPSC)-derived cerebral organoids as a model for recombinant adeno-associated virus (rAAV) capsid selection and for testing efficacy of AAV-based gene therapy in a human context. Cerebral organoids are physiological 3D structures that better recapitulate the human brain compared with 2D cell lines. To validate the model, we compared the transduction efficiency and distribution of two commonly used AAV serotypes (rAAV5 and rAAV9). In cerebral organoids, transduction with rAAV5 led to higher levels of vector DNA, transgenic mRNA, and protein expression as compared with rAAV9. The superior transduction of rAAV5 was replicated in iPSC-derived neuronal cells. Furthermore, rAAV5-mediated delivery of a human sequence-specific engineered microRNA to cerebral organoids led to a lower expression of its target ataxin-3. Our studies provide a new tool for selecting and deselecting AAV serotypes, and for demonstrating therapeutic efficacy of transgenes in a human context. Implementing cerebral organoids during gene therapy development could reduce the usage of animal models and improve translation to the clinic.

AAV Targeting of Glial Cell Types in the Central and Peripheral Nervous System and Relevance to Human Gene Therapy

“…This is an important observation, as it demonstrates findings seen in organoids may recapitulate what would be seen in adult human tissue. Studies have also used human iPSC-derived cerebral organoids to study AAV transduction ( Latour et al, 2019 ; Depla et al, 2020 ). However, these studies only looked at neuronal transgene expression achieved from the AAV vectors used.…”

The Potential of Induced Pluripotent Stem Cells to Test Gene Therapy Approaches for Neuromuscular and Motor Neuron Disorders

“…The transduction properties of two commonly used AAV serotypes (AAV5 and 9) were compared for transgene expression at the mRNA and protein levels, together with the presence of viral DNA. This study reported a higher transduction of the AAV5 compared to AAV9, in organoids and neural cells (Depla et al, 2020). This work set the ground for the use of iPSCs-derived human organoids as valid system for testing AAV properties and will be likely a valuable platform for holistic characterization of AAV properties in vitro and identification of the best therapeutic candidates.…”

Two engineered AAV capsid variants for efficient transduction of human cortical neurons directly converted from iPSC

“…Capsid variant AAV9-NVVRSSS was selected based on a high transduction efficiency in AAV library screens in human cells focusing on the NXXRXXX peptide motif (Börner et al, 2020) (Weinmann et al, unpublished data). By contrast, wildtype AAV2 and AAV9 serotypes poorly transduce human iPSC-derived neurons (Depla et al, 2020; Duong et al, 2019) (Weinmann et al, unpublished data). Cocultures of induced human neurons and primary rat astrocytes in 96-well plates were transduced with AAV vectors encoding an enhanced GFP reporter driven by the ubiquitously active cytomegalovirus promotor at day 21 of maturation.…”

“…Screening of AAV capsid variants in different human primary cell types already pointed to peptide motifs resulting in enhanced transduction rates of specific cell types (Börner et al, 2020; Westhaus et al, 2020). Human neurons differentiated from induced human pluripotent stem cells (iPSC) are increasingly used as a test system for AAV vectors designated for gene therapy of neurological disorders (Depla et al, 2020; Duong et al, 2019). Neuronal differentiation of human iPSC by small molecules however, is a lengthy procedure and leads to a heterogeneous mixture of various neuron types and non-neuronal cells (Strano et al, 2020; Yao et al, 2017).…”