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.

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

Biallelic variations in the () 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 -associated LCA4. In this study, we investigated the potential of adeno-associated virus-mediated gene replacement therapy in two previously validated human retinal organoid (RO) models of LCA4.

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

Mutations in the lebercilin-encoding gene 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 -associated retinal disease. CRISPR-Cas9 technology was used to correct a homozygous nonsense variant in (c.

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.

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 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 mutations.

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 lentiviral transduction of the striatum of Flox-tdTomato Ai9 mice reporter of Cre activity.

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.

CRISPR-Cas9 correction of 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 , a ubiquitously expressed GTPase that regulates mitochondrial homeostasis through coordination of inner membrane fusion, maintenance of cristae structure, and regulation of bioenergetic output.

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.

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.

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.