Exploring the potential of cell-derived vesicles for transient delivery of gene editing payloads.

Gene editing technologies have the potential to correct genetic disorders by modifying, inserting, or deleting specific DNA sequences or genes, paving the way for a new class of genetic therapies. While gene editing tools continue to be improved to increase their precision and efficiency, the limited efficacy of in vivo delivery remains a major hurdle for clinical use. An ideal delivery vehicle should be able to target a sufficient number of diseased cells in a transient time window to maximize on-target editing and mitigate off-target events and immunogenicity. Here, we review major advances in novel delivery platforms based on cell-derived vesicles - extracellular vesicles and virus-like particles - for transient delivery of gene editing payloads. We discuss major findings regarding packaging, in vivo biodistribution, therapeutic efficacy, and safety concerns of cell-derived vesicles delivery of gene-editing cargos and their potential for clinical translation.

Effective AAV-mediated gene replacement therapy in retinal organoids modeling AIPL1-associated LCA4.

Biallelic variations in the aryl hydrocarbon receptor interacting protein-like 1 (AIPL1) gene cause Leber congenital amaurosis subtype 4 (LCA4), an autosomal recessive early-onset severe retinal dystrophy that leads to the rapid degeneration of retinal photoreceptors and the severe impairment of sight within the first few years of life. Currently, there is no treatment or cure for AIPL1-associated LCA4. In this study, we investigated the potential of adeno-associated virus-mediated AIPL1 gene replacement therapy in two previously validated human retinal organoid (RO) models of LCA4. We report here that photoreceptor-specific AIPL1 gene replacement therapy, currently being tested in a first-in-human application, effectively rescued molecular features of AIPL1-associated LCA4 in these models. Notably, the loss of retinal phosphodiesterase 6 was rescued and elevated cyclic guanosine monophosphate (cGMP) levels were reduced following treatment. Transcriptomic analysis of untreated and AAV-transduced ROs revealed transcriptomic changes in response to elevated cGMP levels and viral infection, respectively. Overall, this study supports AIPL1 gene therapy as a promising therapeutic intervention for LCA4.

A Novel Genetic Variant in MBD5 Associated with Severe Epilepsy and Intellectual Disability: Potential Implications on Neural Primary Cilia.

Disruptions in the MBD5 gene have been linked with an array of clinical features such as global developmental delay, intellectual disability, autistic-like symptoms, and seizures, through unclear mechanisms. MBD5 haploinsufficiency has been associated with the disruption of primary cilium-related processes during early cortical development, and this has been reported in many neurodevelopmental disorders. In this study, we describe the clinical history of a 12-year-old child harboring a novel MBD5 rare variant and presenting psychomotor delay and seizures. To investigate the impact of MBD5 haploinsufficiency on neural primary cilia, we established a novel patient-derived cell line and used CRISPR-Cas9 technology to create an isogenic control. The patient-derived neural progenitor cells revealed a decrease in the length of primary cilia and in the total number of ciliated cells. This study paves the way to understanding the impact of MBD5 haploinsufficiency in brain development through its potential impact on neural primary cilia.

CRISPR-Cas9 correction of a nonsense mutation in LCA5 rescues lebercilin expression and localization in human retinal organoids.

Mutations in the lebercilin-encoding gene LCA5 cause one of the most severe forms of Leber congenital amaurosis, an early-onset retinal disease that results in severe visual impairment. Here, we report on the generation of a patient-specific cellular model to study LCA5-associated retinal disease. CRISPR-Cas9 technology was used to correct a homozygous nonsense variant in LCA5 (c.835C>T; p.Q279∗) in patient-derived induced pluripotent stem cells (iPSCs). The absence of off-target editing in gene-corrected (isogenic) control iPSCs was demonstrated by whole-genome sequencing. We differentiated the patient, gene-corrected, and unrelated control iPSCs into three-dimensional retina-like cells, so-called retinal organoids. We observed opsin and rhodopsin mislocalization to the outer nuclear layer in patient-derived but not in the gene-corrected or unrelated control organoids. We also confirmed the rescue of lebercilin expression and localization along the ciliary axoneme within the gene-corrected organoids. Here, we show the potential of combining precise single-nucleotide gene editing with the iPSC-derived retinal organoid system for the generation of a cellular model of early-onset retinal disease.

Extracellular communication between brain cells through functional transfer of Cre mRNA mediated by extracellular vesicles.

In the central nervous system (CNS), the crosstalk between neural cells is mediated by extracellular mechanisms, including brain-derived extracellular vesicles (bdEVs). To study endogenous communication across the brain and periphery, we explored Cre-mediated DNA recombination to permanently record the functional uptake of bdEVs cargo over time. To elucidate functional cargo transfer within the brain at physiological levels, we promoted the continuous secretion of physiological levels of neural bdEVs containing Cre mRNA from a localized region in the brain by in situ lentiviral transduction of the striatum of Flox-tdTomato Ai9 mice reporter of Cre activity. Our approach efficiently detected in vivo transfer of functional events mediated by physiological levels of endogenous bdEVs throughout the brain. Remarkably, a spatial gradient of persistent tdTomato expression was observed along the whole brain, exhibiting an increment of more than 10-fold over 4 months. Moreover, bdEVs containing Cre mRNA were detected in the bloodstream and extracted from brain tissue to further confirm their functional delivery of Cre mRNA in a novel and highly sensitive Nanoluc reporter system. Overall, we report a sensitive method to track bdEV transfer at physiological levels, which will shed light on the role of bdEVs in neural communication within the brain and beyond.

Retinal Organoids from an AIPL1 CRISPR/Cas9 Knockout Cell Line Successfully Recapitulate the Molecular Features of LCA4 Disease.

Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is expressed in photoreceptors where it facilitates the assembly of phosphodiesterase 6 (PDE6) which hydrolyses cGMP within the phototransduction cascade. Genetic variations in AIPL1 cause type 4 Leber congenital amaurosis (LCA4), which presents as rapid loss of vision in early childhood. Limited in vitro LCA4 models are available, and these rely on patient-derived cells harbouring patient-specific AIPL1 mutations. While valuable, the use and scalability of individual patient-derived LCA4 models may be limited by ethical considerations, access to patient samples and prohibitive costs. To model the functional consequences of patient-independent AIPL1 mutations, CRISPR/Cas9 was implemented to produce an isogenic induced pluripotent stem cell line harbouring a frameshift mutation in the first exon of AIPL1. Retinal organoids were generated using these cells, which retained AIPL1 gene transcription, but AIPL1 protein was undetectable. AIPL1 knockout resulted in a decrease in rod photoreceptor-specific PDE6α and ß, and increased cGMP levels, suggesting downstream dysregulation of the phototransduction cascade. The retinal model described here provides a novel platform to assess functional consequences of AIPL1 silencing and measure the rescue of molecular features by potential therapeutic approaches targeting mutation-independent pathogenesis.

Extracellular communication between brain cells through functional transfer of Cre mRNA.

In the central nervous system (CNS), the crosstalk between neural cells is mediated by extracellular mechanisms, including brain-derived extracellular vesicles (bdEVs). To study endogenous communication across the brain and periphery, we explored Cre-mediated DNA recombination to permanently record the functional uptake of bdEVs cargo overtime. To elucidate functional cargo transfer within the brain at physiological levels, we promoted the continuous secretion of physiological levels of neural bdEVs containing Cre mRNA from a localized region in the brain by in situ lentiviral transduction of the striatum of Flox-tdTomato Ai9 mice reporter of Cre activity. Our approach efficiently detected in vivo transfer of functional events mediated by physiological levels of endogenous bdEVs throughout the brain. Remarkably, a spatial gradient of persistent tdTomato expression was observed along the whole brain exhibiting an increment of more than 10-fold over 4 months. Moreover, bdEVs containing Cre mRNA were detected in the bloodstream and extracted from brain tissue to further confirm their functional delivery of Cre mRNA in a novel and highly sensitive Nanoluc reporter system. Overall, we report a sensitive method to track bdEVs transfer at physiological levels which will shed light on the role of bdEVs in neural communication within the brain and beyond.

Induced Pluripotent Stem Cells and Genome-Editing Tools in Determining Gene Function and Therapy for Inherited Retinal Disorders.

Inherited retinal disorders (IRDs) affect millions of people worldwide and are a major cause of irreversible blindness. Therapies based on drugs, gene augmentation or transplantation approaches have been widely investigated and proposed. Among gene therapies for retinal degenerative diseases, the fast-evolving genome-editing CRISPR/Cas technology has emerged as a new potential treatment. The CRISPR/Cas system has been developed as a powerful genome-editing tool in ophthalmic studies and has been applied not only to gain proof of principle for gene therapies in vivo, but has also been extensively used in basic research to model diseases-in-a-dish. Indeed, the CRISPR/Cas technology has been exploited to genetically modify human induced pluripotent stem cells (iPSCs) to model retinal disorders in vitro, to test in vitro drugs and therapies and to provide a cell source for autologous transplantation. In this review, we will focus on the technological advances in iPSC-based cellular reprogramming and gene editing technologies to create human in vitro models that accurately recapitulate IRD mechanisms towards the development of treatments for retinal degenerative diseases.

Investigation of PTC124-mediated translational readthrough in a retinal organoid model of AIPL1-associated Leber congenital amaurosis.

Leber congenital amaurosis type 4 (LCA4), caused by AIPL1 mutations, is characterized by severe sight impairment in infancy and rapidly progressing degeneration of photoreceptor cells. We generated retinal organoids using induced pluripotent stem cells (iPSCs) from renal epithelial cells obtained from four children with AIPL1 nonsense mutations. iPSC-derived photoreceptors exhibited the molecular hallmarks of LCA4, including undetectable AIPL1 and rod cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE6) compared with control or CRISPR-corrected organoids. Increased levels of cGMP were detected. The translational readthrough-inducing drug (TRID) PTC124 was investigated as a potential therapeutic agent. LCA4 retinal organoids exhibited low levels of rescue of full-length AIPL1. However, this was insufficient to fully restore PDE6 in photoreceptors and reduce cGMP. LCA4 retinal organoids are a valuable platform for in vitro investigation of novel therapeutic agents.

CRISPR-Cas9 correction of OPA1 c.1334G>A: p.R445H restores mitochondrial homeostasis in dominant optic atrophy patient-derived iPSCs.

Autosomal dominant optic atrophy (DOA) is the most common inherited optic neuropathy in the United Kingdom. DOA has an insidious onset in early childhood, typically presenting with bilateral, central visual loss caused by the preferential loss of retinal ganglion cells. 60%-70% of genetically confirmed DOA cases are associated with variants in OPA1, a ubiquitously expressed GTPase that regulates mitochondrial homeostasis through coordination of inner membrane fusion, maintenance of cristae structure, and regulation of bioenergetic output. Whether genetic correction of OPA1 pathogenic variants can alleviate disease-associated phenotypes remains unknown. Here, we demonstrate generation of patient-derived OPA1 c.1334G>A: p.R445H mutant induced pluripotent stem cells (iPSCs), followed by correction of OPA1 through CRISPR-Cas9-guided homology-directed repair (HDR) and evaluate the effect of OPA1 correction on mitochondrial homeostasis. CRISPR-Cas9 gene editing demonstrated an efficient method of OPA1 correction, with successful gene correction in 57% of isolated iPSCs. Correction of OPA1 restored mitochondrial homeostasis, re-establishing the mitochondrial network and basal respiration and ATP production levels. In addition, correction of OPA1 re-established the levels of wild-type (WT) mitochondrial DNA (mtDNA) and reduced susceptibility to apoptotic stimuli. These data demonstrate that nuclear gene correction can restore mitochondrial homeostasis and improve mtDNA integrity in DOA patient-derived cells carrying an OPA1 variant.

Protein Delivery of Cell-Penetrating Zinc-Finger Activators Stimulates Latent HIV-1-Infected Cells.

Despite efforts to develop effective treatments for eradicating HIV-1, a cure has not yet been achieved. Whereas antiretroviral drugs target an actively replicating virus, latent, nonreplicative forms persist during treatment. Pharmacological strategies that reactivate latent HIV-1 and expose cellular reservoirs to antiretroviral therapy and the host immune system have, so far, been unsuccessful, often triggering severe side effects, mainly due to systemic immune activation. Here, we present an alternative approach for stimulating latent HIV-1 expression via direct protein delivery of cell-penetrating zinc-finger activators (ZFAs). Cys2-His2 zinc-fingers, fused to a transcription activation domain, were engineered to recognize the HIV-1 promoter and induce targeted viral transcription. Following conjugation with multiple positively charged nuclear localization signal (NLS) repeats, protein delivery of a single ZFA (3NLS-PBS1-VP64) efficiently internalized HIV-1 latently infected T-lymphocytes and specifically stimulated viral expression. We show that short-term treatment with this ZFA protein induces higher levels of viral reactivation in cell line models of HIV-1 latency than those observed with gene delivery. Our work establishes protein delivery of ZFA as a novel and safe approach toward eradication of HIV-1 reservoirs.

Inhibition of HIV replication through siRNA carried by CXCR4-targeted chimeric nanobody.

Small interfering RNA (siRNA) application in therapy still faces a major challenge with the lack of an efficient and specific delivery system. Current vehicles are often responsible for poor efficacy, safety concerns, and burden costs of siRNA-based therapeutics. Here, we describe a novel strategy for targeted delivery of siRNA molecules to inhibit human immunodeficiency virus (HIV) infection. Specific membrane translocation of siRNA inhibitor was addressed by an engineered nanobody targeting the HIV co-receptor CXCR4 (NbCXCR4) in fusion with a single-chain variable fragment (4M5.3) that carried the FITC-conjugated siRNA. 4M5.3-NbCXCR4 conjugate (4M5.3X4) efficiently targeted CXCR4+ T lymphocytes, specifically translocating siRNA by receptor-mediated endocytosis. Targeted delivery of siRNA directed to the mRNA of HIV transactivator tat silenced Tat-driven viral transcription and inhibited the replication of distinct virus clades. In summary, we have shown that the engineered nanobody chimera developed in this study constitutes an efficient and specific delivery method of siRNAs through CXCR4 receptor.

Gene and Cell Therapy for AIPL1-Associated Leber Congenital Amaurosis: Challenges and Prospects.

Leber congenital amaurosis (LCA) caused by AIPL1 mutations is one of the most severe forms of inherited retinal degeneration (IRD). The rapid and extensive photoreceptor degeneration challenges the development of potential treatments. Nevertheless, preclinical studies show that both gene augmentation and photoreceptor transplantation can regenerate and restore retinal function in animal models of AIPL1-associated LCA. However, questions regarding long-term benefit and safety still remain as these therapies advance towards clinical application. Ground-breaking advances in stem cell technology and genome editing are examples of alternative therapeutic approaches and address some of the limitations associated with previous methods. The continuous development of these cutting-edge biotechnologies paves the way towards a bright future not only for AIPL1-associated LCA patients but also other forms of IRD.

Reactivation of Latent HIV-1 Expression by Engineered TALE Transcription Factors.

The presence of replication-competent HIV-1 -which resides mainly in resting CD4+ T cells--is a major hurdle to its eradication. While pharmacological approaches have been useful for inducing the expression of this latent population of virus, they have been unable to purge HIV-1 from all its reservoirs. Additionally, many of these strategies have been associated with adverse effects, underscoring the need for alternative approaches capable of reactivating viral expression. Here we show that engineered transcriptional modulators based on customizable transcription activator-like effector (TALE) proteins can induce gene expression from the HIV-1 long terminal repeat promoter, and that combinations of TALE transcription factors can synergistically reactivate latent viral expression in cell line models of HIV-1 latency. We further show that complementing TALE transcription factors with Vorinostat, a histone deacetylase inhibitor, enhances HIV-1 expression in latency models. Collectively, these findings demonstrate that TALE transcription factors are a potentially effective alternative to current pharmacological routes for reactivating latent virus and that combining synthetic transcriptional activators with histone deacetylase inhibitors could lead to the development of improved therapies for latent HIV-1 infection.

Erythrina mulungu alkaloids are potent inhibitors of neuronal nicotinic receptor currents in mammalian cells.

Crude extracts and three isolated alkaloids from Erythrina mulungu plants have shown anxiolytic effects in different animal models. We investigated whether these alkaloids could affect nicotinic acetylcholine receptors and if they are selective for different central nervous system (CNS) subtypes. Screening experiments were performed using a single concentration of the alkaloid co-applied with acetylcholine in whole cell patch-clamp recordings in three different cell models: (i) PC12 cells natively expressing α3* nicotinic acetylcholine receptors; (ii) cultured hippocampal neurons natively expressing α7* nicotinic acetylcholine receptors; and (iii) HEK 293 cells heterologoulsy expressing α4ß2 nicotinic acetylcholine receptors. For all three receptors, the percent inhibition of acetylcholine-activated currents by (+)-11á-hydroxyerysotrine was the lowest, whereas (+)-erythravine and (+)-11á-hydroxyerythravine inhibited the currents to a greater extent. For the latter two substances, we obtained concentration-response curves with a pre-application protocol for the α7* and α4ß2 nicotinic acetylcholine receptors. The IC50 obtained with (+)-erythravine and (+)-11á-hydroxyerythravine were 6 µM and 5 µM for the α7* receptors, and 13 nM and 4 nM for the α4ß2 receptors, respectively. Our data suggest that these Erythrina alkaloids may exert their behavioral effects through inhibition of CNS nicotinic acetylcholine receptors, particularly the α4ß2 subtype.

Astrocyte-induced synaptogenesis is mediated by transforming growth factor ß signaling through modulation of D-serine levels in cerebral cortex neurons.

Assembly of synapses requires proper coordination between pre- and postsynaptic elements. Identification of cellular and molecular events in synapse formation and maintenance is a key step to understand human perception, learning, memory, and cognition. A key role for astrocytes in synapse formation and function has been proposed. Here, we show that transforming growth factor ß (TGF-ß) signaling is a novel synaptogenic pathway for cortical neurons induced by murine and human astrocytes. By combining gain and loss of function approaches, we show that TGF-ß1 induces the formation of functional synapses in mice. Further, TGF-ß1-induced synaptogenesis involves neuronal activity and secretion of the co-agonist of the NMDA receptor, D-serine. Manipulation of D-serine signaling, by either genetic or pharmacological inhibition, prevented the TGF-ß1 synaptogenic effect. Our data show a novel molecular mechanism that might impact synaptic function and emphasize the evolutionary aspect of the synaptogenic property of astrocytes, thus shedding light on new potential therapeutic targets for synaptic deficit diseases.

Altered oxygen metabolism associated to neurogenesis of induced pluripotent stem cells derived from a schizophrenic patient.

Schizophrenia has been defined as a neurodevelopmental disease that causes changes in the process of thoughts, perceptions, and emotions, usually leading to a mental deterioration and affective blunting. Studies have shown altered cell respiration and oxidative stress response in schizophrenia; however, most of the knowledge has been acquired from postmortem brain analyses or from nonneural cells. Here we describe that neural cells, derived from induced pluripotent stem cells generated from skin fibroblasts of a schizophrenic patient, presented a twofold increase in extramitochondrial oxygen consumption as well as elevated levels of reactive oxygen species (ROS), when compared to controls. This difference in ROS levels was reverted by the mood stabilizer valproic acid. Our model shows evidence that metabolic changes occurring during neurogenesis are associated with schizophrenia, contributing to a better understanding of the development of the disease and highlighting potential targets for treatment and drug screening.

The magnitude of alpha7 nicotinic receptor currents in rat hippocampal neurons is dependent upon GABAergic activity and depolarization.

Hippocampal alpha7(*) nicotinic acetylcholine receptors modulate the release of GABA and glutamate. The control of functional receptor pools by cell firing or synaptic activity could therefore allow for a local adjustment of the sensitivity to cholinergic input upon changes in neuronal activity. We first investigated whether tonic depolarization or cell firing affected the function of alpha7(*). The amplitude of alpha7(*)-gated whole-cell currents in cultured rat hippocampal neurons exposed to high-extracellular K(+) (40 mM KCl) for 24 to 48 h increased 1.3 to 5.5 times. The proportion of alpha7(*)-responsive neurons (99%), the potency of acetylcholine, and the sensitivity to nicotinic antagonists were all unaffected. In contrast, block of spontaneous cell firing with tetrodotoxin for 24 h led to a 37% reduction in mean current amplitude. Reduced alpha7(*) responses were seen after a 24-h blockade of N-type calcium channels but not of L-type calcium channels, N-methyl-d-aspartate (NMDA), or non-NMDA receptor channels, protein kinase C, or calcium-calmodulin kinases II and IV. The N-type or L-type calcium channel antagonists omega-conotoxin GVIA and nifedipine did not prevent the current-potentiating effect of KCl. The GABA(A) antagonist picrotoxin led to a 44% reduction of the currents, despite increasing action potential firing, and also reversed the potentiating effect of KCl. Treatment with GABA, midazolam, or a GABA uptake blocker led to increased currents. These data indicate that alpha7(*)-gated currents in hippocampal neurons are regulated by GABAergic activity and suggest that depolarization-induced GABA release may underlie the effect of increased extracellular KCl.