Abstract Realizing the promise of prime editing for the study and treatment of genetic disorders requires efficient methods for delivering prime editors (PEs) in vivo. Here we describe the identification of bottlenecks limiting adeno-associated virus (AAV)-mediated prime editing in vivo and the development of AAV-PE vectors with increased PE expression, prime editing guide RNA stability and modulation of DNA repair. The resulting dual-AAV systems, v1em and v3em PE-AAV, enable therapeutically relevant prime editing in mouse brain (up to 42% efficiency in cortex), liver (up to 46%) and heart (up to 11%). We apply these systems to install putative protective mutations in vivo for Alzheimer’s disease in astrocytes and for coronary artery disease in hepatocytes. In vivo prime editing with v3em PE-AAV caused no detectable off-target effects or significant changes in liver enzymes or histology. Optimized PE-AAV systems support the highest unenriched levels of in vivo prime editing reported to date, facilitating the study and potential treatment of diseases with a genetic component. 

/pdf/efficient-prime-editing-in-mouse-brain-liver-and-heart-with-2glq7l34.pdf

Efficient prime editing in mouse brain, liver and heart with dual AAVs

Prime editing enables precise installation of genomic substitutions, insertions and deletions in living systems. Efficient in vitro and in vivo delivery of prime editing components, however, remains a challenge. Here we report prime editor engineered virus-like particles (PE-eVLPs) that deliver prime editor proteins, prime editing guide RNAs and nicking single guide RNAs as transient ribonucleoprotein complexes. We systematically engineered v3 and v3b PE-eVLPs with 65- to 170-fold higher editing efficiency in human cells compared to a PE-eVLP construct based on our previously reported base editor eVLP architecture. In two mouse models of genetic blindness, single injections of v3 PE-eVLPs resulted in therapeutically relevant levels of prime editing in the retina, protein expression restoration and partial visual function rescue. Optimized PE-eVLPs support transient in vivo delivery of prime editor ribonucleoproteins, enhancing the potential safety of prime editing by reducing off-target editing and obviating the possibility of oncogenic transgene integration. 

Engineered virus-like particles for transient delivery of prime editor ribonucleoprotein complexes in vivo.

Abstract Genome-editing technologies have ushered in a new era in gene therapy, providing novel therapeutic strategies for a wide range of diseases, including both genetic and nongenetic ocular diseases. These technologies offer new hope for patients suffering from previously untreatable conditions. The unique anatomical and physiological features of the eye, including its immune-privileged status, size, and compartmentalized structure, provide an optimal environment for the application of these cutting-edge technologies. Moreover, the development of various delivery methods has facilitated the efficient and targeted administration of genome engineering tools designed to correct specific ocular tissues. Additionally, advancements in noninvasive ocular imaging techniques and electroretinography have enabled real-time monitoring of therapeutic efficacy and safety. Herein, we discuss the discovery and development of genome-editing technologies, their application to ocular diseases from the anterior segment to the posterior segment, current limitations encountered in translating these technologies into clinical practice, and ongoing research endeavors aimed at overcoming these challenges. 

Genome editing in the treatment of ocular diseases

Recent years have witnessed unprecedented progress in therapeutic gene editing, revolutionizing the approach to treating genetic disorders. In this comprehensive review, we discuss the progression of milestones leading to the emergence of the clustered regularly interspaced short palindromic repeats (CRISPR)-based technology as a powerful tool for precise and targeted modifications of the human genome. CRISPR-Cas9 nuclease, base editing, and prime editing have taken center stage, demonstrating remarkable precision and efficacy in targeted ex vivo and in vivo genomic modifications. Enhanced delivery systems, including viral vectors and nanoparticles, have further improved the efficiency and safety of therapeutic gene editing, advancing their clinical translatability. The exploration of CRISPR-Cas systems beyond the commonly used Cas9, such as the development of Cas12 and Cas13 variants, has expanded the repertoire of gene editing tools, enabling more intricate modifications and therapeutic interventions. Outstandingly, prime editing represents a significant leap forward, given its unparalleled versatility and minimization of off-target effects. These innovations have paved the way for therapeutic gene editing in a multitude of previously incurable genetic disorders, ranging from monogenic diseases to complex polygenic conditions. This review highlights the latest innovative studies in the field, emphasizing breakthrough technologies in preclinical and clinical trials, and their applications in the realm of precision medicine. However, challenges such as off-target effects and ethical considerations remain, necessitating continued research to refine safety profiles and ethical frameworks.

Recent Therapeutic Gene Editing Applications to Genetic Disorders

Inherited retinal diseases (IRDs) encompass a diverse group of genetic disorders that lead to progressive visual impairment and blindness. Over the years, considerable strides have been made in understanding the underlying molecular mechanisms of IRDs, laying the foundation for novel therapeutic interventions. Gene therapy has emerged as a compelling approach for treating IRDs, with notable advancements achieved through targeted gene augmentation. However, several setbacks and limitations persist, hindering the widespread clinical success of gene therapy for IRDs. One promising avenue of research is the development of new genome editing tools. Cutting-edge technologies such as CRISPR-Cas9 nucleases, base editing and prime editing provide unprecedented precision and efficiency in targeted gene manipulation, offering the potential to overcome existing challenges in gene therapy for IRDs. Furthermore, traditional gene therapy encounters a significant challenge due to immune responses to viral vectors, which remain crucial obstacles in achieving long-lasting therapeutic effects. Nanotechnology has emerged as a valuable ally in the quest to optimize gene therapy outcomes for ocular diseases. Nanoparticles engineered with nanoscale precision offer improved gene delivery to specific retinal cells, allowing for enhanced targeting and reduced immunogenicity. In this review, we discuss recent advancements in gene therapy for IRDs and explore the setbacks that have been encountered in clinical trials. We highlight the technological advances in genome editing for the treatment of IRDs and how integrating nanotechnology into gene delivery strategies could enhance the safety and efficacy of gene therapy, ultimately offering hope for patients with IRDs and potentially paving the way for similar advancements in other ocular disorders.

Gene therapy for inherited retinal diseases: exploiting new tools in genome editing and nanotechnology

Retinitis pigmentosa (RP) is an inherited retinal dystrophy causing progressive and irreversible loss of retinal photoreceptors. Here, we developed a genome-editing tool characterized by the versatility of prime editors (PEs) and unconstrained PAM requirement of a SpCas9 variant (SpRY), referred to as PESpRY. The diseased retinas of Pde6b-associated RP mouse model were transduced via a dual AAV system packaging PESpRY for the in vivo genome editing through a non-NGG PAM (GTG). The progressing cell loss was reversed once the mutation was corrected, leading to substantial rescue of photoreceptors and production of functional PDE6β. The treated mice exhibited significant responses in electroretinogram and displayed good performance in both passive and active avoidance tests. Moreover, they presented an apparent improvement in visual stimuli-driven optomotor responses and efficiently completed visually guided water-maze tasks. Together, our study provides convincing evidence for the prevention of vision loss caused by RP-associated gene mutations via unconstrained in vivo prime editing in the degenerating retinas.

Vision rescue via unconstrained in vivo prime editing in degenerating neural retinas.

Color blindness is a retinal disease that mainly manifests as a color vision disorder, characterized by achromatopsia, red-green color blindness, and blue-yellow color blindness. With the development of technology and progress in theory, extensive research has been conducted on the genetic basis of color blindness, and various approaches have been explored for its treatment. This article aims to provide a comprehensive review of recent advances in understanding the pathological mechanism, clinical symptoms, and treatment options for color blindness. Additionally, we discuss the various treatment approaches that have been developed to address color blindness, including gene therapy, pharmacological interventions, and visual aids. Furthermore, we highlight the promising results from clinical trials of these treatments, as well as the ongoing challenges that must be addressed to achieve effective and long-lasting therapeutic outcomes. Overall, this review provides valuable insights into the current state of research on color blindness, with the intention of informing further investigation and development of effective treatments for this disease.

Dyschromatopsia: a comprehensive analysis of mechanisms and cutting-edge treatments for color vision deficiency

X-linked retinitis pigmentosa, primarily caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene, represents one of the most severe forms of inherited retinal degeneration, with early onset and rapid progression. Conventional interventions, such as vitamin A or docosahexaenoic acid supplementation, offer limited benefits and fail to halt disease progression. By contrast, gene therapy has emerged as a promising approach to alter the disease course. The present review summarizes the clinical phenotypes and pathogenic mechanisms associated with RPGR mutations, focusing on their disruption of ciliary transport and metabolic homeostasis. The present review further discusses advances in preclinical models, including mice, dogs, zebrafish and induced pluripotent stem cell-derived organoids, that have facilitated the development of RPGR-targeted therapies. Adeno-associated virus-based gene replacement has shown efficacy in restoring retinal structure and function, and several approaches have progressed to early-phase clinical trials. Despite encouraging outcomes, challenges such as RPGR coding sequence instability, vector delivery efficiency and long-term safety remain. The present review integrates current mechanistic understanding and therapeutic progress, providing a translational perspective for precision treatment of RPGR-associated retinal diseases.

Advances in RPGR gene therapy for X-linked retinitis pigmentosa: From preclinical insights to clinical application (Review)

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Vision rescue via unconstrained in vivo prime editing in degenerating neural retinas.

Dyschromatopsia: a comprehensive analysis of mechanisms and cutting-edge treatments for color vision deficiency

Advances in RPGR gene therapy for X-linked retinitis pigmentosa: From preclinical insights to clinical application (Review)