Recent Advances in CRISPR-Cas9 Delivery Methods for In Vivo Gene Therapy

ABSTRACT Introduction: Therapeutic gene editing is becoming a viable biomedical tool with the emergence of the CRISPR/Cas9 system. CRISPR-based technologies have promise as a therapeutic platform for many human genetic diseases previously considered untreatable, providing a flexible approach to high-fidelity gene editing. For many diseases, such as sickle-cell disease and beta thalassemia, curative therapy may already be on the horizon, with CRISPR-based clinical trials slated for the next few years. Translation of CRISPR-based therapy to in vivo application however, is no small feat, and major hurdles remain for efficacious use of the CRISPR/Cas9 system in clinical contexts. Areas covered: In this topical review, we highlight recent advances to in vivo delivery of the CRISPR/Cas9 system using various packaging formats, including viral, mRNA, plasmid, and protein-based approaches. We also discuss some of the barriers which have yet to be overcome for successful translation of this technology. Expert opinion: This review focuses on the challenges to efficacy for various delivery formats, with specific emphasis on overcoming these challenges through the development of carrier vehicles for transient approaches to CRISPR/Cas9 delivery in vivo.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has revolutionized the field of gene editing. Continuous efforts in developing this technology have enabled efficient in vitro, ex vivo, and in vivo gene editing through a variety of delivery strategies. Viral vectors are commonly used in in vitro, ex vivo, and in vivo delivery systems, but they can cause insertional mutagenesis, have limited cloning capacity, and/or elicit immunologic responses. Physical delivery methods are largely restricted to in vitro and ex vivo systems, whereas chemical delivery methods require extensive optimization to improve their efficiency for in vivo gene editing. Achieving a safe and efficient in vivo delivery system for CRISPR/Cas9 remains the most challenging aspect of gene editing. Recently, extracellular vesicle-based systems were reported in various studies to deliver Cas9 in vitro and in vivo. In comparison with other methods, extracellular vesicles offer a safe, transient, and cost-effective yet efficient platform for delivery, indicating their potential for Cas9 delivery in clinical trials. In this review, we first discuss the pros and cons of different Cas9 delivery strategies. We then specifically review the development of extracellular vesicle-mediated gene editing and highlight the strengths and weaknesses of this technology.

… far, the effective delivery of multiple CRISPR components in vivo into host cells still … vivo therapeutic CRISPR delivery platforms and address some of the challenges of CRISPR delivery…

Within less than a decade since its inception, CRISPR-Cas9-based genome editing has been rapidly advanced to human clinical trials in multiple disease areas. Although it is highly anticipated that this revolutionary technology will bring novel therapeutic modalities to many diseases by precisely manipulating cellular DNA sequences, the low efficiency of in vivo delivery must be enhanced before its therapeutic potential can be fully realized. Here we discuss the most recent progress of in vivo delivery of CRISPR-Cas9 systems, highlight innovative viral and non-viral delivery technologies, emphasize outstanding delivery challenges, and provide the most updated perspectives.

… To test whether CRISPR/Cas9 components delivered by our … For in vivo test, one injection of Cas9 mRNA LLNs followed … system can effectively deliver CRISPR/Cas9 components to …

Owing to its easy-to-use and multiplexing nature, the genome editing tool CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease 9) is revolutionizing many areas of medical research and one of the most amazing areas is its gene therapy potentials. Previous explorations into the therapeutic potentials of CRISPR-Cas9 were mainly conducted in vitro or in animal germlines, the translatability of which, however, is either limited (to tissues with adult stem cells amenable to culture and manipulation) or currently impermissible (due to ethic concerns). Recently, important progresses have been made on this regard. Several studies have demonstrated the ability of CRISPR-Cas9 for in vivo gene therapy in adult rodent models of human genetic diseases delivered by methods that are potentially translatable to human use. Although these recent advances represent a significant step forward to the eventual application of CRISPR-Cas9 to the clinic, there are still many hurdles to overcome, such as the off-target effects of CRISPR-Cas9, efficacy of homology-directed repair, fitness of edited cells, immunogenicity of therapeutic CRISPR-Cas9 components, as well as efficiency, specificity, and translatability of in vivo delivery methods. In this article, we introduce the mechanisms and merits of CRISPR-Cas9 in genome editing, briefly retrospect the applications of CRISPR-Cas9 in gene therapy explorations and highlight recent advances, later we discuss in detail the challenges lying ahead in the way of its translatability, propose possible solutions, and future research directions.

Therapies that target the function of immune cells have significant clinical efficacy in diseases such as cancer and autoimmunity. Although functional genomics has accelerated therapeutic target discovery in cancer, its use in primary immune cells is limited because vector delivery is inefficient and can perturb cell states. Here we describe CHIME: CHimeric IMmune Editing, a CRISPR-Cas9 bone marrow delivery system to rapidly evaluate gene function in innate and adaptive immune cells in vivo without ex vivo manipulation of these mature lineages. This approach enables efficient deletion of genes of interest in major immune lineages without altering their development or function. We use this approach to perform an in vivo pooled genetic screen and identify Ptpn2 as a negative regulator of CD8+ T cell-mediated responses to LCMV Clone 13 viral infection. These findings indicate that this genetic platform can enable rapid target discovery through pooled screening in immune cells in vivo. The use of functional genomics in primary immune cells has been limited by inefficient vector delivery and risk of perturbing cell states. Here the authors present CHimeric IMmune Editing (CHIME) for in vivo evaluation of gene function and pooled screening approaches.

… , proper delivery of its components is crucial. Both viral and nonviral delivery methods are … In addition, we review ex vivo and in vivo CRISPR/Cas9 applications for disease therapies. …

ABSTRACT Introduction Gene therapy is becoming increasingly common in clinical practice, giving hope for the correction of a wide range of human diseases and defects. The CRISPR/Cas9 system, consisting of the Cas9 nuclease and single-guide RNA (sgRNA), has revolutionized the field of gene editing. However, efficiently delivering the CRISPR-Cas9 to the target organ or cell remains a significant challenge. In recent years, with rapid advances in nanoscience, materials science, and medicine, researchers have developed various technologies that can deliver CRISPR-Cas9 in different forms for in vitro and in vivo gene editing. Here, we review the development of the CRISPR-Cas9 and describe the delivery forms and the vectors that have emerged in CRISPR-Cas9 delivery, summarizing the key barriers and the promising strategies that vectors currently face in delivering the CRISPR-Cas9. Areas covered With the rapid development of CRISPR-Cas9, delivery methods are becoming increasingly important in the in vivo delivery of CRISPR-Cas9. Expert opinion CRISPR-Cas9 is becoming increasingly common in clinical trials. However, the complex nuclease and protease environment is a tremendous challenge for in vivo clinical applications. Therefore, the development of delivery methods is highly likely to take the application of CRISPR-Cas9 technology to another level.

Clustered randomly interspaced short palindromic repeats (CRISPRs) and its associated endonuclease protein, i.e., Cas9, have been discovered as an immune system in bacteria and archaea; nevertheless, they are now being adopted as mainstream biotechnological/molecular scissors that can modulate ample genetic and nongenetic diseases via insertion/deletion, epigenome editing, messenger RNA editing, CRISPR interference, etc. Many Food and Drug Administration‐approved and ongoing clinical trials on CRISPR adopt ex vivo strategies, wherein the gene editing is performed ex vivo, followed by reimplantation to the patients. However, the in vivo delivery of the CRISPR components is still under preclinical surveillance. This review has summarized the nonviral nanodelivery strategies for gene editing using CRISPR/Cas9 and its recent advancements, strategic points of view, challenges, and future aspects for tissue‐specific in vivo delivery of CRISPR/Cas9 components using nanomaterials.

The development of clinically viable delivery methods presents one of the greatest challenges in the therapeutic application of CRISPR/Cas9 mediated genome editing. Here, we report the development of a lipid nanoparticle (LNP)-mediated delivery system that, with a single administration, enabled significant editing of the mouse transthyretin (Ttr) gene in the liver, with a >97% reduction in serum protein levels that persisted for at least 12 months. These results were achieved with an LNP delivery system that was biodegradable and well tolerated. The LNP delivery system was combined with a sgRNA having a chemical modification pattern that was important for high levels of in vivo activity. The formulation was similarly effective in a rat model. Our work demonstrates that this LNP system can deliver CRISPR/Cas9 components to achieve clinically relevant levels of in vivo genome editing with a concomitant reduction of TTR serum protein, highlighting the potential of this system as an effective genome editing platform.

The discovery that the bacterial defense mechanism, CRISPR-Cas9, can be reprogrammed as a gene editing tool has revolutionized the field of gene editing. CRISPR-Cas9 can introduce a double-strand break at a specific targeted site within the genome. Subsequent intracellular repair mechanisms repair the double strand break that can either lead to gene knock-out (via the non-homologous end-joining pathway) or specific gene correction in the presence of a DNA template via homology-directed repair. With the latter, pathological mutations can be cut out and repaired. Advances are being made to utilize CRISPR-Cas9 in patients by incorporating its components into non-viral delivery vehicles that will protect them from premature degradation and deliver them to the targeted tissues. Herein, CRISPR-Cas9 can be delivered in the form of three different cargos: plasmid DNA, RNA or a ribonucleoprotein complex (RNP). We and others have recently shown that Cas9 RNP can be efficiently formulated in lipid-nanoparticles (LNP) leading to functional delivery in vitro. In this study, we compared LNP encapsulating the mRNA Cas9, sgRNA and HDR template against LNP containing Cas9-RNP and HDR template. Former showed smaller particle sizes, better protection against degrading enzymes and higher gene editing efficiencies on both reporter HEK293T cells and HEPA 1-6 cells in in vitro assays. Both formulations were additionally tested in female Ai9 mice on biodistribution and gene editing efficiency after systemic administration. LNP delivering mRNA Cas9 were retained mainly in the liver, with LNP delivering Cas9-RNPs additionally found in the spleen and lungs. Finally, gene editing in mice could only be concluded for LNP delivering mRNA Cas9 and sgRNA. These LNPs resulted in 60 % gene knock-out in hepatocytes. Delivery of mRNA Cas9 as cargo format was thereby concluded to surpass Cas9-RNP for application of CRISPR-Cas9 for gene editing in vitro and in vivo.

Since its mechanism discovery in 2012 and the first application for mammalian genome editing in 2013, CRISPR-Cas9 has revolutionized the genome engineering field and created countless opportunities in both basic science and translational medicine. The first clinical trial of CRISPR therapeutics was initiated in 2016, which employed ex vivo CRISPR-Cas9 edited PD-1 knockout T cells for the treatment of non-small cell lung cancer. So far there have been dozens of clinical trials registered on ClinicalTrials.gov in regard to using the CRISPR-Cas9 genome editing as the main intervention for therapeutic applications; however, most of these studies use ex vivo genome editing approach, and only a few apply the in vivo editing strategy. Compared to ex vivo editing, in vivo genome editing bypasses tedious procedures related to cell isolation, maintenance, selection, and transplantation. It is also applicable to a wide range of diseases and disorders. The main obstacles to the successful translation of in vivo therapeutic genome editing include the lack of safe and efficient delivery system and safety concerns resulting from the off-target effects. In this review, we highlight the therapeutic applications of in vivo genome editing mediated by the CRISPR-Cas9 system. Following a brief introduction of the history, biology, and functionality of CRISPR-Cas9, we showcase a series of exemplary studies in regard to the design and implementation of in vivo genome editing systems that target the brain, inner ear, eye, heart, liver, lung, muscle, skin, immune system, and tumor. Current challenges and opportunities in the field of CRISPR-enabled therapeutic in vivo genome editing are also discussed.

The potential of clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR associated protein 9 (Cas9)-based therapeutic genome editing is hampered by difficulties in the control of the in vivo activity of CRISPR–Cas9. To minimize any genotoxicity, precise activation of CRISPR–Cas9 in the target tissue is desirable. Here, we show that, by complexing magnetic nanoparticles with recombinant baculoviral vectors (MNP-BVs), CRISPR–Cas9-mediated genome editing can be activated locally in vivo via a magnetic field. The baculoviral vector was chosen for in vivo gene delivery because of its large loading capacity and ability to locally overcome systemic inactivation by the complement system. We demonstrate that a locally applied magnetic field can enhance the cellular entry of MNP-BVs, thereby avoiding baculoviral vector inactivation and causing a transient transgene expression in the target tissue. Because baculoviral vectors are inactivated elsewhere, gene delivery and in vivo genome editing via MNP-BVs are tissue specific. CRISPR–Cas9-mediated genome editing can be activated locally in vivo via an applied magnetic field, after complexation of magnetic nanoparticles with recombinant baculoviral vectors packaging the CRISPR–Cas9 machinery.

Chronic hepatitis B virus (HBV) infection constitutes a global health issue with limited current therapeutic efficacy owing to the persistence of viral episomal DNA (cccDNA). The CRISPR/Cas9 system, a newly developed, powerful tool for genome editing and potential gene therapy, requires efficient delivery of CRISPR components for successful therapeutic application. Here, we investigated the effects of lentiviral- or adeno-associated virus 2 (AAV2) vector-mediated delivery of 3 guide (g)RNAs/Cas9 selected from 16 gRNAs. These significantly suppressed HBV replication in cells, with WJ11/Cas9 exhibiting highest efficacy and chosen for in vivo study. AAV2/WJ11-Cas9 also significantly inhibited HBV replication and significantly reduced cccDNA in the tested cells. Moreover, AAV2/WJ11-Cas9 enhanced entecavir effects when used in combination, indicative of different modes of action. Notably, in humanized chimeric mice, AAV2/WJ11-Cas9 significantly suppressed HBcAg, HBsAg, and HBV DNA along with cccDNA in the liver tissues without significant cytotoxicity; accordingly, next generation sequencing data showed no significant genomic mutations. To our knowledge, this represents the first evaluation of the CRISPR/Cas9 system using an HBV natural infection mode. Therefore, WJ11/Cas9 delivered by comparatively safer AAV2 vectors may provide a new therapeutic strategy for eliminating HBV infection and serve as an effective platform for curing chronic HBV infection.

Hemophilia is a hereditary disease that remains incurable. Although innovative treatments such as gene therapy or bispecific antibody therapy have been introduced, substantial unmet needs still exist with respect to achieving long-lasting therapeutic effects and treatment options for inhibitor patients. Antithrombin (AT), an endogenous negative regulator of thrombin generation, is a potent genome editing target for sustainable treatment of patients with hemophilia A and B. In this study, we developed and optimized lipid nanoparticles (LNPs) to deliver Cas9 mRNA along with single guide RNA that targeted AT in the mouse liver. The LNP-mediated CRISPR-Cas9 delivery resulted in the inhibition of AT that led to improvement in thrombin generation. Bleeding-associated phenotypes were recovered in both hemophilia A and B mice. No active off-targets, liver-induced toxicity, and substantial anti-Cas9 immune responses were detected, indicating that the LNP-mediated CRISPR-Cas9 delivery was a safe and efficient approach for hemophilia therapy.

A main challenge to broaden the biomedical application of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9) genome editing technique is the delivery of Cas9 nuclease and single‐guide RNA (sgRNA) into the specific cell and organ. An effective and very fast CRISPR/Cas9 genome editing in vitro and in vivo enabled by bioreducible lipid/Cas9 messenger RNA (mRNA) nanoparticle is reported. BAMEA‐O16B, a lipid nanoparticle integrated with disulfide bonds, can efficiently deliver Cas9 mRNA and sgRNA into cells while releasing RNA in response to the reductive intracellular environment for genome editing as fast as 24 h post mRNA delivery. It is demonstrated that the simultaneous delivery of Cas9 mRNA and sgRNA using BAMEA‐O16B knocks out green fluorescent protein (GFP) expression of human embryonic kidney cells with efficiency up to 90%. Moreover, the intravenous injection of BAMEA‐O16B/Cas9 mRNA/sgRNA nanoparticle effectively accumulates in hepatocytes, and knocks down proprotein convertase subtilisin/kexin type 9 level in mouse serum down to 20% of nontreatment. The leading lipid nanoparticle, BAMEA‐O16B, represents one of the most efficient CRISPR/Cas9 delivery nanocarriers reported so far, and it can broaden the therapeutic promise of mRNA and CRISPR/Cas9 technique further.

… For instance, folate-linked lipoplexes for targeted delivery of … on the delivery of the CRISPR/Cas9 system in vivo using the … apply this vector to deliver the CRISPR/Cas9 system in …

… trials, in vivo application is still restricted by the instability and inefficient delivery of CRISPR–Cas9 … In this Review, we summarize ex vivo and in vivo strategies to deliver CRISPR–Cas9 …

The complexity of CRISPR machinery is a challenge to its application for nonviral in vivo therapeutic gene editing. Here, we demonstrate that proteins, regardless of size or charge, efficiently load into porous silicon nanoparticles (PSiNPs). Optimizing the loading strategy yields formulations that are ultrahigh loading─>40% cargo by volume─and highly active. Further tuning of a polymeric coating on the loaded PSiNPs yields nanocomposites that achieve colloidal stability under cryopreservation, endosome escape, and gene editing efficiencies twice that of the commercial standard Lipofectamine CRISPRMAX. In a mouse model of arthritis, PSiNPs edit cells in both the cartilage and synovium of knee joints, and achieve 60% reduction in expression of the therapeutically relevant MMP13 gene. Administered intramuscularly, they are active over a broad dose range, with the highest tested dose yielding nearly 100% muscle fiber editing at the injection site. The nanocomposite PSiNPs are also amenable to systemic delivery. Administered intravenously in a model that mimics muscular dystrophy, they edit sites of inflamed muscle. Collectively, the results demonstrate that the PSiNP nanocomposites are a versatile system that can achieve high loading of diverse cargoes and can be applied for gene editing in both local and systemic delivery applications.

Genetic diseases seriously threaten human health and have always been one of the refractory conditions facing humanity. Currently, gene therapy drugs such as siRNA, shRNA, antisense oligonucleotide, CRISPR/Cas9 system, plasmid DNA and miRNA have shown great potential in biomedical applications. To avoid the degradation of gene therapy drugs in the body and effectively deliver them to target tissues, cells and organelles, the development of excellent drug delivery vehicles is of utmost importance. Viral vectors are the most widely used delivery vehicles for gene therapy in vivo and in vitro due to their high transfection efficiency and stable transgene expression. With the development of nanotechnology, novel nanocarriers are gradually replacing viral vectors, emerging superior performance. This review mainly illuminates the current widely used gene therapy drugs, summarizes the viral vectors and non-viral vectors that deliver gene therapy drugs, and sums up the application of gene therapy to treat genetic diseases. Additionally, the challenges and opportunities of the field are discussed from the perspective of developing an effective nano-delivery system.

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system is a gene-editing technology. Nanoparticle delivery systems have attracted attention because of the limitations of conventional viral vectors. In this review, we assess the efficiency of various nanoparticles, including lipid-based, polymer-based, inorganic, and extracellular vesicle-based systems, as non-viral vectors for CRISPR/Cas9 delivery. We discuss their advantages, limitations, and current challenges. By summarizing recent advancements and highlighting key strategies, this review aims to provide a comprehensive overview of the role of non-viral delivery systems in advancing CRISPR/Cas9 technology for clinical applications and gene therapy.

The CRISPR-Cas9 technology, one of the groundbreaking genome editing methods for addressing genetic disorders, has emerged as a powerful, precise, and efficient tool. However, its clinical translation remains hindered by challenges in delivery efficiency and targeting specificity. This review provides a comprehensive analysis of the structural features, advantages, and potential applications of various non-viral and stimuli-responsive systems, examining recent progress to emphasize the potential to address these limitations and advance CRISPR-Cas9 therapeutics. We describe how recent reports emphasize that nonviral vectors, including lipid-based nanoparticles, extracellular vesicles, polymeric nanoparticles, gold nanoparticles, and mesoporous silica nanoparticles, can offer diverse advantages to enhance stability, cellular uptake, and biocompatibility, based on their structures and physio-chemical stability. We also summarize recent progress on stimuli-responsive nanoformulations, a type of non-viral vector, to introduce precision and control in CRISPR-Cas9 delivery. Stimuli-responsive nanoformulations are designed to respond to pH, redox states, and external triggers, facilitate controlled and targeted delivery, and minimize off-target effects. The insights in our review suggest future challenges for clinical applications of gene therapy technologies and highlight the potential of delivery systems to enhance CRISPR-Cas9’s clinical efficacy, positioning them as pivotal tools for future gene-editing therapies.

Clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has revolutionized the field of gene therapy as it has enabled precise genome editing with unprecedented accuracy and efficiency, paving the way for clinical applications to treat otherwise incurable genetic disorders. Typically, precise genome editing requires the delivery of multiple components to the target cells that, depending on the editing platform used, may include messenger RNA (mRNA), protein complexes, and DNA fragments. For clinical purposes, these have to be efficiently delivered into transplantable cells, such as primary T lymphocytes or hematopoietic stem and progenitor cells that are typically sensitive to exogenous substances. This challenge has limited the broad applicability of precise gene therapy applications to those strategies for which efficient delivery methods are available. Electroporation-based methodologies have been generally applied for gene editing applications, but procedure-associated toxicity has represented a major burden. With the advent of novel and less disruptive methodologies to deliver genetic cargo to transplantable cells, it is now possible to safely and efficiently deliver multiple components for precise genome editing, thus expanding the applicability of these strategies. In this review, we describe the different delivery systems available for genome editing components, including viral and non-viral systems, highlighting their advantages, limitations, and recent clinical applications. Recent improvements to these delivery methods to achieve cell specificity represent a critical development that may enable in vivo targeting in the future and will certainly play a pivotal role in the gene therapy field.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system adapted from bacteria is a programmable nuclease-based genome editing tool. The long-lasting effect of gene silencing or correction is beneficial in cancer treatment. Considering the need to broaden the practical application of this technology, highly efficient non-viral vectors are urgently required. We prepared a multifunctional non-viral vector that could actively target tumor cells and deliver CRISPR/Cas9 plasmids into nuclei of cancer cells. Protamine sulfate (PS) which contains nuclear localization sequence was utilized to condense plasmid DNA and facilitate nuclei-targeted delivery. Liposome-coated protein/DNA complex avoided the degradation of nuclease in blood circulation. The obtained PS@Lip/pCas9 was further modified with distearoyl phosphoethanolamine-polyethylene glycol-hyaluronic acid (HA) to endow the vector ability to actively target tumor cell. Results suggested that PS@HA-Lip could deliver CRISPR/Cas9 plasmids into nuclei of tumor cells and induce genome editing effect. With the disruption of MTH1 (mutT homolog1) gene, the growth of non-small cell lung cancer was inhibited. Moreover, cell apoptosis in tumor tissue was promoted, and liver metastasis of non-small cell lung cancer (NSCLC) was reduced. Our study has provided a therapeutic strategy targeting MTH1 gene for NSCLC therapy. STATEMENT OF SIGNIFICANCE: CRISPR/Cas9 as a powerful tool for genome editing has drawn much attention. The long-lasting effect possesses unique advantage in cancer treatment. Non-viral vectors have high loading capacity, high safety and low immunogenicity, playing an important role in CRISPR/Cas9 delivery. In our study, a multifunctional non-viral vector for the efficient delivery of CRISPR/Cas9 plasmid was constructed. With the active targeting ligand and nuclei-targeting component, the cargo was efficiently delivered into cell nuclei and exerted genome editing effect. By using this vector, we successfully inhibited the growth and induced the apoptosis of non-small cell lung cancer by disrupting MTH1 expression with good safety. Our work provided an efficient non-vial vector for CRISPR/Cas9 delivery and explored the possibility for cancer treatment.

Abstract CRISPR/Cas is a revolutionary gene editing technology with wide‐ranging utility. The safe, non‐viral delivery of CRISPR/Cas components would greatly improve future therapeutic utility. We report the synthesis and development of zwitterionic amino lipids (ZALs) that are uniquely able to (co)deliver long RNAs including Cas9 mRNA and sgRNAs. ZAL nanoparticle (ZNP) delivery of low sgRNA doses (15 n m ) reduces protein expression by >90 % in cells. In contrast to transient therapies (such as RNAi), we show that ZNP delivery of sgRNA enables permanent DNA editing with an indefinitely sustained 95 % decrease in protein expression. ZNP delivery of mRNA results in high protein expression at low doses in vitro (<600 pM) and in vivo (1 mg kg −1 ). Intravenous co‐delivery of Cas9 mRNA and sgLoxP induced expression of floxed tdTomato in the liver, kidneys, and lungs of engineered mice. ZNPs provide a chemical guide for rational design of long RNA carriers, and represent a promising step towards improving the safety and utility of gene editing.

CRISPR–Cas9 is a revolutionary genome-editing technology that has enormous potential for the treatment of genetic diseases. However, the lack of efficient and safe, non-viral delivery …

Genome editing, especially via the simple and versatile type II CRISPR/Cas9 system, offers an effective avenue to precisely control cell fate, an important aspect of tissue regeneration. Unfortunately, most CRISPR/Cas9 non-viral delivery strategies only utilise micro-/nano-particle delivery methods. While these approaches provide reasonable genomic editing efficiencies, their systemic delivery may lead to undesirable off-target effects. For in vivo applications, a more localized and sustained delivery approach may be useful, particularly in the context of tissue regeneration. Here, we developed a scaffold that delivers the CRISPR/Cas9 components (i.e. single guide RNA (sgRNA) and Cas9 protein complexes) in a localized and non-viral manner. Specifically, using mussel-inspired bioadhesive coating, polyDOPA-melanin (pDOPA), we adsorbed Cas9:sgRNA lipofectamine complexes onto bio-mimicking fiber scaffolds. To evaluate the genome-editing efficiency of this platform, U2OS.EGFP cells were used as the model cell type. pDOPA coating was essential in allowing Cas9:sgRNA lipofectamine complexes to adhere onto the scaffolds with a higher loading efficiency, while laminin coating was necessary for maintaining cell viability and proliferation on the pDOPA-coated fibers for effective gene editing (21.5% editing efficiency, p < 0.001). Importantly, U2OS.EGFP cells took up Cas9:sgRNA lipofectamine complexes directly from the scaffolds via reverse transfection. Overall, we demonstrate the efficacy of such fiber scaffolds in providing localized, sustained and non-viral delivery of Cas9:sgRNA complexes. Such genome editing scaffolds may find useful applications in tissue regeneration. STATEMENT OF SIGNIFICANCE: Currently, there is a lack of effective non-viral means to deliver CRISPR/Cas9 components for genome editing. Most existing approaches only utilize micro-/nano-particles by injection or systemic delivery, which may lead to undesirable off-target effects. Here, we report a platform that delivers the CRISPR/Cas9 components (i.e. single guide RNA (sgRNA) and Cas9 protein complexes) in a localized and sustained manner. We used mussel-inspired bioadhesive coating to functionalize the bio-mimicking fiber scaffolds with Cas9:sgRNA lipofectamine complexes, to allow effective gene editing for the cells seeded on the scaffolds. Importantly, the cells took up Cas9:sgRNA lipofectamine complexes directly from the scaffolds. Such genome editing scaffolds may find useful applications in tissue regeneration.

… 9 (CRISPR/Cas9)-… , delivering the CRISPR/Cas9 system effectively to the liver for in vivo gene editing of AS is still a challenge. Herein, we reported a non-viral CRISPR/Cas9 delivery …

Recent advances in molecular biology have led to the CRISPR revolution, but the lack of an efficient and safe delivery system into cells and tissues continues to hinder clinical translation of CRISPR approaches. Polymeric vectors offer an attractive alternative to viruses as delivery vectors due to their large packaging capacity and safety profile. In this paper, we have demonstrated the potential use of a highly branched poly(β-amino ester) polymer, HPAE-EB, to enable genomic editing via CRISPRCas9-targeted genomic excision of exon 80 in the COL7A1 gene, through a dual-guide RNA sequence system. The biophysical properties of HPAE-EB were screened in a human embryonic 293 cell line (HEK293), to elucidate optimal conditions for efficient and cytocompatible delivery of a DNA construct encoding Cas9 along with two RNA guides, obtaining 15–20% target genomic excision. When translated to human recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes, transfection efficiency and targeted genomic excision dropped. However, upon delivery of CRISPR–Cas9 as a ribonucleoprotein complex, targeted genomic deletion of exon 80 was increased to over 40%. Our study provides renewed perspective for the further development of polymer delivery systems for application in the gene editing field in general, and specifically for the treatment of RDEB.

Clustered regulatory interspaced short palindromic repeats or CRISPR/Cas9 has emerged as a potent and versatile tool for efficient genome editing. This technology has been exploited for several applications including disease modelling, cell therapy, diagnosis, and treatment of many diseases including cancer. The in vivo application of CRISPR/Cas9 is hindered by poor stability, pharmacokinetic profile, and the limited ability of the CRISPR payloads to cross biological barriers. Although viral vectors have been implemented as delivery tools for efficient in vivo gene editing, their application is associated with high immunogenicity and toxicity, limiting their clinical translation. Hence, there is a need to explore new delivery methods that can guarantee safe and efficient delivery of the CRISPR/Cas9 components to target cells. In this review, we first provide a brief history and principles of nuclease-mediated gene editing, we then focus on the different CRISPR/Cas9 formats outlining their potentials and limitations. Finally, we discuss the alternative non-viral delivery strategies currently adopted for in vivo CRISPR/Cas9 gene editing.

Genome-editing technologies such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENS), and the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein system have revolutionized biological research. Each biotechnology consists of a DNA-binding protein that can be programmed to recognize and initiate double-strand breaks (DSBs) for site-specific gene modification. These technologies have the potential to be harnessed to cure diseases caused by aberrant gene expression. To be successful therapeutically, their functionality depends on their safe and efficient delivery into the cell nucleus. This review discusses the challenges in the delivery of genome-editing tools, and highlights recent innovations in non-viral delivery that have potential to overcome these limitations and advance the translation of genome editing towards patient care.

The combination of Cas9, guide RNA and repair template DNA can induce precise gene editing and the correction of genetic diseases in adult mammals. However, clinical implementation of this technology requires safe and effective delivery of all of these components into the nuclei of the target tissue. Here, we combine lipid nanoparticle–mediated delivery of Cas9 mRNA with adeno-associated viruses encoding a sgRNA and a repair template to induce repair of a disease gene in adult animals. We applied our delivery strategy to a mouse model of human hereditary tyrosinemia and show that the treatment generated fumarylacetoacetate hydrolase (Fah)-positive hepatocytes by correcting the causative Fah-splicing mutation. Treatment rescued disease symptoms such as weight loss and liver damage. The efficiency of correction was >6% of hepatocytes after a single application, suggesting potential utility of Cas9-based therapeutic genome editing for a range of diseases.

… on non-viral delivery of genome-editing nucleases. In particular, we focus on non-viral delivery of Cas9/… In addition, the future direction for developing non-viral delivery of programmable …

Background/Objectives: While CRISPR/Cas9 technology offers a revolutionary approach for correcting genetic ocular blindness, efficient and safe delivery remains the primary bottleneck. Traditional viral vectors, despite their efficacy, face challenges regarding cargo size limitations and potential genomic integration risks. Non-viral vectors offer distinct comparative advantages, including large cargo capacity for diverse CRISPR tools and transient expression to minimize off-target effects, but must overcome the eye’s formidable static and dynamic barriers, specifically the corneal epithelium, vitreous humor, and the inner limiting membrane. In this review, we present an anatomically guided framework for non-viral CRISPR/Cas9 delivery, mapping engineering strategies to specific ocular tissue targets. We first delineate the mechanisms of key physiological barriers, including the corneal stroma, aqueous humor circulation, and the vitreous–retina interface. Subsequently, we critically evaluate the latest advancements in non-viral platforms, such as pH-responsive lipid nanoparticles and engineered virus-like particles. The core focus of this review is on site-specific breakthrough strategies: from utilizing mucoadhesive polymers to counteract tear clearance in the cornea to exploiting specialized administration routes, such as suprachoroidal space and subretinal injection, to bypass retinal barriers, and deep-penetrating intravitreal carriers for targeting the photoreceptor-RPE complex. By integrating material science with precise administration routes, this review highlights feasible translational pathways for next-generation, carrier-free, or biomimetic ocular gene editing therapies.

… lot of importance in CRIPSPR-Cas9 delivery. The nanocarrier … field of CRISPR-Cas9 using non-viral vectors that are used … limitations of non-viral based CRISPR-Cas9 editing platform …

In recent years, CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) genome editing systems have become one of the most robust platforms in basic biomedical research and therapeutic applications. To date, efficient in vivo delivery of the CRISPR/Cas9 system to the targeted cells remains a challenge. Although viral vectors have been widely used in the delivery of the CRISPR/Cas9 system in vitro and in vivo, their fundamental shortcomings, such as the risk of carcinogenesis, limited insertion size, immune responses and difficulty in large-scale production, severely limit their further applications. Alternative non-viral delivery systems for CRISPR/Cas9 are urgently needed. With the rapid development of non-viral vectors, lipid- or polymer-based nanocarriers have shown great potential for CRISPR/Cas9 delivery. In this review, we analyze the pros and cons of delivering CRISPR/Cas9 systems in the form of plasmid, mRNA, or protein and then discuss the limitations and challenges of CRISPR/Cas9-based genome editing. Furthermore, current non-viral vectors that have been applied for CRISPR/Cas9 delivery in vitro and in vivo are outlined in details. Finally, critical obstacles for non-viral delivery of CRISPR/Cas9 system are highlighted and promising strategies to overcome these barriers are proposed.

Since its discovery, the CRISPR/Cas technology has rapidly become an essential tool in modern biomedical research. The opportunities to specifically modify and correct genomic DNA have also raised big hope for therapeutic applications by direct in vivo genome editing. In order to achieve the intended genome modifications, the functional unit of the CRISPR/Cas system finally has to be present in the nucleus of target cells. This can be achieved by delivery of different biomolecular Cas9 and gRNA formats: plasmid DNA (pDNA), RNA or Cas9 ribonucleoproteins (RNPs). While the initial research focussed on pDNA transfections, the currently most promising strategy for systemic non-viral in vivo delivery is based on RNA which has achieved remarkable results in the first clinical trials. RNP delivery receives much attention for ex vivo applications, but the translation to systemic in vivo genome editing in patients has not been reached so far. The article summarises the characteristics and differences of each format, provides an overview of the published delivery strategies and highlights recent examples of delivery systems including the status of clinical applications.

In vivo gene editing therapies offer the potential to treat the root causes of many genetic diseases. Realizing the promise of therapeutic in vivo gene editing requires the ability to safely and efficiently deliver gene editing agents to relevant organs and tissues in vivo. Here, we review current delivery technologies that have been used to enable therapeutic in vivo gene editing, including viral vectors, lipid nanoparticles, and virus-like particles. Since no single delivery modality is likely to be appropriate for every possible application, we compare the benefits and drawbacks of each method and highlight opportunities for future improvements.

… gene therapy so we will also discuss in vivo and ex vivo delivery approaches, and delivery of the therapies based on gene editing, … vehicles will be developed, enabling genome editing …

The field of genome engineering has created new possibilities for gene therapy, including improved animal models of disease, engineered cell therapies, and in vivo gene repair. The most significant challenge for the clinical translation of genome engineering is the development of safe and effective delivery vehicles. A large body of work has applied genome engineering to genetic modification in vitro, and clinical trials have begun using cells modified by genome editing. Now, promising preclinical work is beginning to apply these tools in vivo. This article summarizes the development of genome engineering platforms, including meganucleases, zinc finger nucleases, TALENs, and CRISPR/Cas9, and their flexibility for precise genetic modifications. The prospects for the development of safe and effective viral and nonviral delivery vehicles for genome editing are reviewed, and promising advances in particular therapeutic applications are discussed.

CRISPR-based genome editing holds promise for addressing genetic disease, infectious disease, and cancer and has rapidly advanced from primary research to clinical trials in recent years. However, the lack of safe and potent in vivo delivery methods for CRISPR components has limited most ongoing clinical trials to ex vivo gene therapy. Effective CRISPR in vivo genome editing necessitates an effective vehicle ensuring target cell transduction while minimizing off-target effects, toxicity, and immune reactions. In this review, we examine promising biological-derived platforms to deliver DNA editing agents in vivo and the engineering thereof, encompassing potent viral-based vehicles, flexible protein nanocages, and mammalian-derived particles.

… To achieve high efficiency, tissue specificity and safety in in vivo genome editing using engineered delivery vehicles, modelling, simulation and computational analysis can significantly …

Adeno-associated virus (AAV) has shown promising therapeutic efficacy with a good safety profile in a wide range of animal models and human clinical trials. With the advent of clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome-editing technologies, AAV provides one of the most suitable viral vectors to package, deliver, and express CRISPR components for targeted gene editing. Recent discoveries of smaller Cas9 orthologues have enabled the packaging of Cas9 nuclease and its chimeric guide RNA into a single AAV delivery vehicle for robust in vivo genome editing. Here, we discuss how the combined use of small Cas9 orthologues, tissue-specific minimal promoters, AAV serotypes, and different routes of administration has advanced the development of efficient and precise in vivo genome editing and comprehensively review the various AAV-CRISPR systems that have been effectively used in animals. We then discuss the clinical implications and potential strategies to overcome off-target effects, immunogenicity, and toxicity associated with CRISPR components and AAV delivery vehicles. Finally, we discuss ongoing non-viral-based ex vivo gene therapy clinical trials to underscore the current challenges and future prospects of CRISPR/Cas9 delivery for human therapeutics.

Viruses and virally derived particles have the intrinsic capacity to deliver molecules to cells, but the difficulty of readily altering cell-type selectivity has hindered their use for therapeutic delivery. Here, we show that cell surface marker recognition by antibody fragments displayed on membrane-derived particles encapsulating CRISPR–Cas9 protein and guide RNA can deliver genome editing tools to specific cells. Compared to conventional vectors like adeno-associated virus that rely on evolved capsid tropisms to deliver virally encoded cargo, these Cas9-packaging enveloped delivery vehicles (Cas9-EDVs) leverage predictable antibody–antigen interactions to transiently deliver genome editing machinery selectively to cells of interest. Antibody-targeted Cas9-EDVs preferentially confer genome editing in cognate target cells over bystander cells in mixed populations, both ex vivo and in vivo. By using multiplexed targeting molecules to direct delivery to human T cells, Cas9-EDVs enable the generation of genome-edited chimeric antigen receptor T cells in humanized mice, establishing a programmable delivery modality with the potential for widespread therapeutic utility. Cell-specific molecular delivery with enveloped delivery vehicles enables genome editing ex vivo and in vivo.

ConspectusClustered, regularly interspaced, short palindromic repeat (CRISPR)/associated protein 9 (CRISPR/Cas9) is emerging as a powerful genome-editing tool, enabling precise and targeted modifications of virtually any genomic sequence in living cells. These technologies have potential therapeutic applications for cancers, metabolic diseases, and genetic disorders. However, several major challenges hinder the full realization of their potential. Specifically, CRISPR-Cas9 gene editors, whether delivered as plasmid DNA, mRNA/sgRNA, or ribonucleoprotein (RNP), exhibit poor membrane permeability, restricting their access to the intracellular genome, where the editing occurs. Additionally, these editors lack tissue or organ specificity, raising concerns about off-target editing at the tissue level that causes unwanted genotoxicity. Though a range of delivery carriers has been developed to deliver Cas9 editors, their effectiveness is often limited by a number of barriers at both the extracellular and intracellular levels. Moreover, the prolonged activity of Cas9 increases the risk of off-target editing at the genomic level. Therefore, it is crucial to develop efficient delivery vectors, along with molecular switches to safely regulate Cas9 activity.In this Account, we summarize our recent achievements in developing different types of materials that can efficiently deliver the plasmid DNA encoding Cas9 protein and single-guide RNA (sgRNA), or Cas9 RNP into cells to highlight the design considerations of carriers for safe and efficient delivery in vitro and in vivo. After elucidating the chemical and physical factors that are responsible for encapsulating and delivering these biomacromolecules, we further elucidate how we design the biodegradable polymeric carriers using dynamic disulfide chemistry, emphasize their safe and efficient delivery features for genome-editing biomacromolecules, and also introduce the integration of the intracellular delivery of genome-editing biomacromolecules with microneedle-based transdermal delivery to promote therapeutic genome editing for inflammatory skin disorders. Finally, we review how we exploit optical, chemical, and genetic switches to control the Cas9 activity in conjunction with targeted delivery to address the spatiotemporal specificity of gene editing in vivo and demonstrate their precision therapy against cancer and colitis treatment as proof-of-concept examples. In the final part, we will summarize the progress we have made and propose the future directions that may impact the field based on our own research outcomes.

Chimeric antigen receptor (CAR) cell therapy has achieved groundbreaking success in treating hematological malignancies. However, its application to solid tumors remains challenging due to complex manufacturing processes, limited in vivo persistence, and transient therapeutic effects. In vivo CAR-immune cells induced by gene delivery systems loaded with CAR genes and gene-editing tools have shown efficiency for anti-tumor immunotherapy. In situ programming of autologous immune cells avoids the safety concerns of allogeneic immune cells, and the manufacture of gene delivery systems could be standardized. Therefore, the in vivo editing and in situ generation of CAR-immune cells might potentially overcome the abovementioned limitations of current CAR cell therapy. This review mainly focuses on CAR structures, gene-editing tools, and gene delivery techniques applied in anti-tumor immunotherapy to help design and develop in situ CAR-immune cell therapy. The recent applications of in vivo CAR-immune cell therapy in both hematologic malignancies and solid tumors are investigated. To sum up, the in vivo editing and in situ generation of CAR therapy holds promise for offering a practical, cost-effective, efficient, safe, and widely applicable approach to the next-generation anti-tumor immunotherapy.

ABSTRACT Mucopolysaccharidosis type I (MPS I) is a multisystemic disorder caused by the deficiency of alpha‐L‐iduronidase (IDUA) that leads to intracellular accumulation of glycosaminoglycans (GAG). In the present study we aimed to use cationic liposomes carrying the CRISPR/Cas9 plasmid and a donor vector for in vitro and in vivo MPS I gene editing, and compare to treatment with naked plasmids. The liposomal formulation was prepared by microfluidization. Complexes were obtained by the addition of DNA at +4/−1 charge ratio. The overall results showed complexes of about 110 nm, with positive zeta potential of +30 mV. The incubation of the complexes with fibroblasts from MPS I patients led to a significant increase in IDUA activity and reduction of lysosomal abnormalities. Hydrodynamic injection of the liposomal complex in newborn MPS I mice led to a significant increase in serum IDUA levels for up to six months. The biodistribution of complexes after hydrodynamic injection was markedly detected in the lungs and heart, corroborating the results of increased IDUA activity and decreased GAG storage especially in these tissues, while the group that received the naked plasmids presented increased enzyme activity especially in the liver. Furthermore, animals treated with the liposomal formulation presented improvement in cardiovascular parameters, one of the main causes of death observed in MPS I patients. We conclude that the IDUA production in multiple organs had a significant beneficial effect on the characteristics of MPS I disease, which may bring hope to gene therapy of Hurler patients. Graphical abstract Figure. No Caption available. HighlightsLiposomes are efficient carriers for CRISPR/Cas9 system.Liposomal CRISPR/Cas9 complexes showed efficient gene editing of MPS I fibroblasts.Treatment of newborn MPS I mice promoted high serum IDUA levels for up to 6 months.Treatment with liposomal complexes was more efficient than Naked CRISPR/Cas9.Liposomal CRISPR/Cas9‐treated mice improved GAG accumulation and cardiac function.

Hematopoietic stem cells (HSCs) provide the body with a continuous supply of healthy, functional blood cells. In patients with hematopoietic malignancies, immunodeficiencies, lysosomal storage disorders, and hemoglobinopathies, therapeutic genome editing offers hope for corrective intervention, with even modest editing efficiencies likely to provide clinical benefit. Engineered white blood cells, such as T cells, can be applied therapeutically to address monogenic disorders of the immune system, HIV infection, or cancer. The versatility of CRISPR-based tools allows countless new medical interventions for diseases of the blood, and rapid ex vivo success has been demonstrated in hemoglobinopathies via transplantation of the patient’s HSCs following genome editing in a laboratory setting. Here we review recent advances in therapeutic genome editing of HSCs and T cells, focusing on the progress in ex vivo contexts, the promise of improved access via in vivo delivery, as well as the ongoing preclinical efforts that may enable the transition from ex vivo to in vivo administration. We discuss the challenges, limitations, and future prospects of this rapidly developing field, which may one day establish CRISPR as the standard of care for some diseases affecting the blood.

… genome editing machinery specifically to target cells, thereby enabling precise and permanent gene editing in predefined genomic … in vivo engineering strategies using delivery vehicles …

Precise gene manipulation by gene editing approaches facilitates the potential to cure several debilitating genetic disorders. Gene modification stimulated by engineered nucleases induces a double-stranded break (DSB) in the target genomic locus, thereby activating DNA repair mechanisms. DSBs triggered by nucleases are repaired either by the nonhomologous end-joining or the homology-directed repair pathway, enabling efficient gene editing. While there are several ongoing ex vivo genome editing clinical trials, current research underscores the therapeutic potential of CRISPR/Cas-based (clustered regularly interspaced short palindrome repeats-associated Cas nuclease) in vivo gene editing. In this review, we provide an overview of the CRISPR/Cas-mediated in vivo genome therapy applications and explore their prospective clinical translatability to treat human monogenic disorders. In addition, we discuss the various challenges associated with in vivo genome editing technologies and strategies used to circumvent them. Despite the robust and precise nuclease-mediated gene editing, a promoterless, nuclease-independent gene targeting strategy has been utilized to evade the drawbacks of the nuclease-dependent system, such as off-target effects, immunogenicity, and cytotoxicity. Thus, the rapidly evolving paradigm of gene editing technologies will continue to foster the progress of gene therapy applications.

CRISPR-Cas9–mediated genome editing can be used to treat ALS in an animal model of the disease. Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease characterized by the progressive loss of motor neurons in the spinal cord and brain. In particular, autosomal dominant mutations in the superoxide dismutase 1 (SOD1) gene are responsible for ~20% of all familial ALS cases. The clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas9) genome editing system holds the potential to treat autosomal dominant disorders by facilitating the introduction of frameshift-induced mutations that can disable mutant gene function. We demonstrate that CRISPR-Cas9 can be harnessed to disrupt mutant SOD1 expression in the G93A-SOD1 mouse model of ALS following in vivo delivery using an adeno-associated virus vector. Genome editing reduced mutant SOD1 protein by >2.5-fold in the lumbar and thoracic spinal cord, resulting in improved motor function and reduced muscle atrophy. Crucially, ALS mice treated by CRISPR-mediated genome editing had ~50% more motor neurons at end stage and displayed a ~37% delay in disease onset and a ~25% increase in survival compared to control animals. Thus, this study illustrates the potential for CRISPR-Cas9 to treat SOD1-linked forms of ALS and other central nervous system disorders caused by autosomal dominant mutations.

Abstract Transient delivery of CRISPR‐based genome editing effectors is important to reduce off‐target effects and immune responses. Recently extracellular vesicles (EVs) have been explored for Cas9 ribonucleoprotein (RNP) delivery. However, lack of mechanisms to enrich RNPs into EVs limited the efficiency of EVs as a RNP delivery vehicle. Here we describe a mechanism to actively enrich RNPs into EVs. We used the specific interaction between RNA aptamer and aptamer‐binding protein (ABP) to enrich RNPs into EVs. We inserted RNA aptamer com into single guide RNA (sgRNA), and fused com‐binding ABP Com to both termini of tetraspan protein CD63 that is abundant in exosomes. We found that the Com/com interaction enriched Cas9 and adenine base editor (ABE) RNPs into EVs, via forming a three‐component complex including CD63‐Com fusion protein, com‐modified sgRNA and Cas9 or ABE. The RNP enriched EVs are efficient in genome editing and transiently expressed. The system is capable of delivering RNPs targeting multiple loci for multiplex genome editing. In addition, Cas9 from different species can be used together. The EV‐delivered RNPs are active in vivo. The data show that the aptamer and ABP interactions can be utilized to actively enrich RNPs into EVs for improved genome editing efficiency and safety.

… vivo genetic engineering to develop a PH1 mouse model and evaluate therapeutic effects of LNP-mediated CRISPR-Cas9 … single administration of LNP-CRISPR-Cas9 targeting mHao1 …

CRISPR/Cas9-based genome-editing therapies are poised to change the clinical outcome for many diseases with validated therapeutic targets awaiting an appropriate delivery system. Recent advances in lipid nanoparticle (LNP) technology make them an attractive platform for the delivery of various forms of CRISPR/Cas9, including the efficient and transient Cas9/gRNA ribonucleoprotein (RNP) complexes. In this study, we initially tested our novel LNP platform by delivering pre-complexed RNPs and template DNA to cultured mouse cortical neurons, and obtained successful ex vivo genome editing. We then directly injected LNP-packaged RNPs and DNA template into the mouse cornea to evaluate in vivo delivery. For the first time, we demonstrated wide-spread genome editing in the cornea using our LNP-RNPs. The ability of our LNPs to transfect the cornea highlights the potential of our novel delivery platform to be used in CRISPR/Cas9-based genome editing therapies of corneal diseases.

In the past 10 years, CRISPR-Cas9 has revolutionized the gene-editing field due to its modularity, simplicity, and efficacy. It has been applied for the creation of in vivo models, to further understand human biology, and toward the curing of genetic diseases. However, there remain significant delivery barriers for CRISPR-Cas9 application in the clinic, especially for in vivo and extrahepatic applications. In this work, high-throughput molecular barcoding techniques were used alongside traditional screening methodologies to simultaneously evaluate LNP formulations encapsulating ribonucleoproteins (RNPs) for in vitro gene-editing efficiency and in vivo biodistribution. This resulted in the identification of a lung-tropic LNP formulation, which shows efficient gene editing in endothelial and epithelial cells within the lung, targeting both model reporter and clinically relevant genomic targets. Further, this LNP shows no off-target indel formation in the liver, making it a highly specific extrahepatic delivery system for lung-editing applications.

Hemophilia is a genetic disorder that is caused by mutations in coagulation factor VIII (hemophilia A) or IX (hemophilia B) genes resulting in blood clotting disorders. Despite advances in therapies, such as recombinant proteins and products with extended half-lives, the treatment of hemophilia still faces two major limitations: the short duration of therapeutic effect and production of neutralizing antibodies against clotting factors (inhibitor). To overcome these limitations, new hemophilia treatment strategies have been established such as gene therapy, bispecific antibody, and rebalancing therapy. Although these strategies have shown promising results, it is difficult to achieve a permanent therapeutic effect. Advances in the clustered regularly interspaced short palindromic repeat (CRISPR) technology have allowed sustainable treatment by correcting mutated genes. Since genome editing generates irreversible changes in host genome, safety must be ensured by delivering target organs. Therefore, the delivery tool of the CRISPR system is crucial for safe, accurate, and efficient genome editing. Recently, non-viral vector lipid nanoparticles (LNPs) have emerged as safer tools for delivering CRISPR systems than other viral vectors. Several previous hemophilia pre-clinical studies using LNP-CRISPR showed that sufficient and sustainable therapeutic effects, which means that LNP-CRISPR-mediated genome-editing therapy can be a valid option for the treatment of hemophilia. In this paper, we summarize the latest advancements in the successful treatment of hemophilia and the potential of CRISPR-mediated genome-editing therapy using LNPs.

Genome editing therapy for Duchenne muscular dystrophy (DMD) holds great promise, however, one major obstacle is delivery of the CRISPR-Cas9/sgRNA system to skeletal muscle tissues. In general, AAV vectors are used for in vivo delivery, but AAV injections cannot be repeated because of neutralization antibodies. Here we report a chemically defined lipid nanoparticle (LNP) system which is able to deliver Cas9 mRNA and sgRNA into skeletal muscle by repeated intramuscular injections. Although the expressions of Cas9 protein and sgRNA were transient, our LNP system could induce stable genomic exon skipping and restore dystrophin protein in a DMD mouse model that harbors a humanized exon sequence. Furthermore, administration of our LNP via limb perfusion method enables to target multiple muscle groups. The repeated administration and low immunogenicity of our LNP system are promising features for a delivery vehicle of CRISPR-Cas9 to treat skeletal muscle disorders. In vivo delivery of CRISPR-Cas9 holds promise for treating muscular dystrophy, however, AAV delivery is known to be immunogenic. Here, the authors show that LNP delivery of CRISPR-Cas9 enables repeated injections into skeletal muscle and leads to restored dystrophin expression in multiple muscle groups.

BACKGROUND Transthyretin amyloidosis, also called ATTR amyloidosis, is a life-threatening disease characterized by progressive accumulation of misfolded transthyretin (TTR) protein in tissues, predominantly the nerves and heart. NTLA-2001 is an in vivo gene-editing therapeutic agent that is designed to treat ATTR amyloidosis by reducing the concentration of TTR in serum. It is based on the clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR-Cas9) system and comprises a lipid nanoparticle encapsulating messenger RNA for Cas9 protein and a single guide RNA targeting TTR. METHODS After conducting preclinical in vitro and in vivo studies, we evaluated the safety and pharmacodynamic effects of single escalating doses of NTLA-2001 in six patients with hereditary ATTR amyloidosis with polyneuropathy, three in each of the two initial dose groups (0.1 mg per kilogram and 0.3 mg per kilogram), within an ongoing phase 1 clinical study. RESULTS Preclinical studies showed durable knockout of TTR after a single dose. Serial assessments of safety during the first 28 days after infusion in patients revealed few adverse events, and those that did occur were mild in grade. Dose-dependent pharmacodynamic effects were observed. At day 28, the mean reduction from baseline in serum TTR protein concentration was 52% (range, 47 to 56) in the group that received a dose of 0.1 mg per kilogram and was 87% (range, 80 to 96) in the group that received a dose of 0.3 mg per kilogram. CONCLUSIONS In a small group of patients with hereditary ATTR amyloidosis with polyneuropathy, administration of NTLA-2001 was associated with only mild adverse events and led to decreases in serum TTR protein concentrations through targeted knockout of TTR. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.).

Lipid nanoparticle (LNP) delivery of clustered regularly interspaced short palindromic repeat (CRISPR) ribonucleoproteins (RNPs) could enable high-efficiency, low-toxicity and scalable in vivo genome editing if efficacious RNP–LNP complexes can be reliably produced. Here we engineer a thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) to generate iGeoCas9 variants capable of >100× more genome editing of cells and organs compared with the native GeoCas9 enzyme. Furthermore, iGeoCas9 RNP–LNP complexes edit a variety of cell types and induce homology-directed repair in cells receiving codelivered single-stranded DNA templates. Using tissue-selective LNP formulations, we observe genome-editing levels of 16‒37% in the liver and lungs of reporter mice that receive single intravenous injections of iGeoCas9 RNP–LNPs. In addition, iGeoCas9 RNPs complexed to biodegradable LNPs edit the disease-causing SFTPC gene in lung tissue with 19% average efficiency, representing a major improvement over genome-editing levels observed previously using viral or nonviral delivery strategies. These results show that thermostable Cas9 RNP–LNP complexes can expand the therapeutic potential of genome editing. An engineered clustered regularly interspaced short palindromic repeat ribonucleoprotein delivered in lipid nanoparticles efficiently edits cells in vivo.

As an important modality for choroidal neovascularization (CNV) treatment, intravitreal injection of vascular endothelial growth factor A (VEGFA) inhibitors suffers from undesired response rate, low patient compliance, and ocular damage. Here, dynamically covalent lipid nanoparticles (LNPs) were engineered to mediate VEGFA gene editing and CNV treatment by codelivering Cas9 mRNA (mCas9) and single guide RNA (sgRNA) targeting VEGFA (sgVEGFA). A library of lipidoids bearing iminoboronate ester linkage was developed via facile “one-pot” synthesis, and the top-performing lipidoid-A4B3C7 was formulated into LNP-A4B3C7 with the highest mRNA transfection efficiency. Inside the diseased retinal pigment epithelial cells, LNPs were dissociated upon H2O2-triggered lipidoid degradation, facilitating mRNA/sgRNA release to potentiate the gene editing efficiency. In laser-induced CNV mice, mCas9/sgVEGFA@LNP-A4B3C7 after single intravitreal injection led to pronounced VEGFA disruption and CNV area reduction, outperforming the clinical anti-VEGF drug in eliciting sustained therapeutic effect. This study establishes a robust nonviral platform for mRNA delivery and genome editing and renders a promising strategy for CNV treatment.

The development of melanoma is closely related to Braf gene, which is a suitable target for CRISPR/Cas9 based gene therapy. CRISPR/Cas9-sgRNA ribonucleoprotein complexes (RNPs) stand out as the safest format compared to plasmid and mRNA delivery. Similarly, lipid nanoparticles (LNPs) emerge as a safer alternative to viral vectors for delivering the CRISPR/Cas9-sgRNA gene editing system. Herein, we have designed multifunctional cationic LNPs specifically tailored for the efficient delivery of Cas9 RNPs targeting the mouse Braf gene through transdermal delivery, aiming to treat mouse melanoma. LNPs are given a positive charge by the addition of a newly synthesized polymer, deoxycholic acid modified polyethyleneimine (PEI-DOCA). Positive charge enables LNPs to be delivered in vivo by binding to negatively charged cell membranes and proteins, thereby facilitating efficient skin penetration and enhancing the delivery of RNPs into melanoma cells for gene editing purposes. Our research demonstrates that these LNPs enhance drug penetration through the skin, successfully delivering the Cas9 RNPs system and specifically targeting the Braf gene. Cas9 RNPs loaded LNPs exert a notable impact on gene editing in melanoma cells, significantly suppressing their proliferation. Furthermore, in mice experiments, the LNPs exhibited skin penetration and tumor targeting capabilities. This innovative LNPs delivery system offers a promising gene therapy approach for melanoma treatment and provides fresh insights into the development of safe and effective delivery systems for Cas9 RNPs in vivo. STATEMENT OF SIGNIFICANCE: CRISPR/Cas9 technology brings new hope for cancer treatment. Cas9 ribonucleoprotein offers direct genome editing, yet delivery challenges persist. For melanoma, transdermal delivery minimizes toxicity but faces skin barrier issues. We designed multifunctional lipid nanoparticles (LNPs) for Cas9 RNP delivery targeting the Braf gene. With metal microneedle pretreatment, our LNPs effectively edited melanoma cells, reducing Braf expression and inhibiting tumor growth. Our study demonstrates LNPs' potential for melanoma therapy and paves the way for efficient in vivo Cas9 RNP delivery systems in cancer therapy.

A scaffold-mediated system was developed to enhance the local delivery of Cas9 complexes for targeting leukemia stem cells in AML. Leukemia stem cells (LSCs) sustain the disease and contribute to relapse in acute myeloid leukemia (AML). Therapies that ablate LSCs may increase the chance of eliminating this cancer in patients. To this end, we used a bioreducible lipidoid-encapsulated Cas9/single guide RNA (sgRNA) ribonucleoprotein [lipidoid nanoparticle (LNP)–Cas9 RNP] to target the critical gene interleukin-1 receptor accessory protein (IL1RAP) in human LSCs. To enhance LSC targeting, we loaded LNP-Cas9 RNP and the chemokine CXCL12α onto mesenchymal stem cell membrane–coated nanofibril (MSCM-NF) scaffolds mimicking the bone marrow microenvironment. In vitro, CXCL12α release induced migration of LSCs to the scaffolds, and LNP-Cas9 RNP induced efficient gene editing. IL1RAP knockout reduced LSC colony-forming capacity and leukemic burden. Scaffold-based delivery increased the retention time of LNP-Cas9 in the bone marrow cavity. Overall, sustained local delivery of Cas9/IL1RAP sgRNA via CXCL12α-loaded LNP/MSCM-NF scaffolds provides an effective strategy for attenuating LSC growth to improve AML therapy.

In vitro and ex-vivo target identification strategies often fail to predict in vivo efficacy, particularly for glioblastoma (GBM), a highly heterogenous tumor rich in resistant cancer stem cells (GSCs). An in vivo screening tool can improve prediction of therapeutic efficacy by considering the complex tumor microenvironment and the dynamic plasticity of GSCs driving therapy resistance and recurrence. This study proposes lipid nanoparticles (LNPs) as an efficient in vivo CRISPR-Cas9 gene editing tool for target validation in mesenchymal GSCs. LNPs co-delivering mRNA (mCas9) and single-guide RNA (sgRNA) were successfully formulated and optimized facilitating both in vitro and in vivo gene editing. In vitro, LNPs achieved up to 67 % reduction in green fluorescent protein (GFP) expression, used as a model target, outperforming a commercial transfection reagent. Intratumoral administration of LNPs in GSCs resulted in ~80 % GFP gene knock-out and a 2-fold reduction in GFP signal by day 14. This study showcases the applicability of CRISPR-Cas9 LNPs as a potential in vivo screening tool in GSCs, currently lacking effective treatment. By replacing GFP with a pool of potential targets, the proposed platform presents an exciting prospect for therapeutic target validation in orthotopic GSCs, bridging the gap between preclinical and clinical research.

Lipid tail heterogeneity enables organ-selective mRNA delivery for in vivo CRISPR-Cas9 gene editing - ScienceDirect …

Lipid nanoparticles (LNPs) enable efficient mRNA delivery, yet their potential for ocular gene editing remains largely unexplored. Here, we systematically evaluated three LNP formulations containing distinct ionizable lipids, DLin-MC3-DMA, ALC0315, and SM102, for gene delivery to ocular tissues. Among them, SM102-based LNP encapsulating GFP mRNA (SM102-GFP) exhibited the highest transfection efficiency across three cultured ocular cells in vitro. Following intravitreal injection in mice, SM102-GFP achieved selective and robust expression in the trabecular meshwork (TM) without detectable retinal transfection. GFP expression in TM peaked at one week post-injection, declined by three weeks, and could be effectively re-induced by a second dosing of the same vector. Compared with adeno-associated viral (AAV) and adenoviral (Ad) vectors, SM102-GFP showed superior TM specificity and reduced retinal inflammation. Co-delivery of SpCas9 mRNA and sgRNA via SM102-based LNPs enabled efficient CRISPR-mediated knockout of Matrix Gla Protein (Mgp), a key inhibitor of TM calcification. Mgp knockout induced sustained intraocular pressure elevation and anterior chamber deepening with open angles, recapitulating features of primary open-angle glaucoma. Chronic ocular hypertension further led to Müller gliosis and ganglion cell complex thinning, indicative of progressive retinal stress. These findings establish SM102-based LNPs as a safe and efficient platform for TM-targeted gene editing and glaucoma modeling.

Oral treatment of colon diseases with the CRISPR/Cas9 system has been hampered by the lack of a safe and efficient delivery platform. Overexpressed CD98 plays a crucial role in the progression of ulcerative colitis (UC) and colitis-associated colorectal cancer (CAC). In this study, lipid nanoparticles (LNPs) derived from mulberry leaves are functionalized with Pluronic copolymers and optimized to deliver the CRISPR/Cas gene editing machinery for CD98 knockdown. The obtained LNPs possessed a hydrodynamic diameter of 267.2 nm, a narrow size distribution, and a negative surface charge (-25.6 mV). Incorporating Pluronic F127 into LNPs improved their stability in the gastrointestinal tract and facilitated their penetration through the colonic mucus barrier. The galactose end groups promoted endocytosis of the LNPs by macrophages via asialoglycoprotein receptor-mediated endocytosis, with a transfection efficiency of 2.2-fold higher than Lipofectamine 6000. The LNPs significantly decreased CD98 expression, down-regulated pro-inflammatory cytokines (TNF-α and IL-6), up-regulated anti-inflammatory factors (IL-10), and polarized macrophages to M2 phenotype. Oral administration of LNPs mitigated UC and CAC by alleviating inflammation, restoring the colonic barrier, and modulating intestinal microbiota. As the first oral CRISPR/Cas9 delivery LNP, this system offers a precise and efficient platform for the oral treatment of colon diseases.

The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-based biotechnologies has revolutionized the life sciences and introduced new therapeutic modalities with the potential to treat a wide range of diseases. Here, we describe CRISPR-based strategies to improve human health, with an emphasis on the delivery of CRISPR therapeutics directly into the human body using adeno-associated virus (AAV) vectors. We also discuss challenges facing broad deployment of CRISPR-based therapeutics and highlight areas where continued discovery and technological development can further advance these revolutionary new treatments.

The adeno-associated virus (AAV) is a defective single-stranded DNA virus with the simplest structure reported to date. It constitutes a capsid protein and single-stranded DNA. With its high transduction efficiency, low immunogenicity, and tissue specificity, it is the most widely used and promising gene therapy vector. The clustered regularly interspaced short palindromic sequence (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing system is an emerging technology that utilizes cas9 nuclease to specifically recognize and cleave target genes under the guidance of small guide RNA and realizes gene editing through homologous directional repair and non-homologous recombination repair. In recent years, an increasing number of animal experiments and clinical studies have revealed the great potential of AAV as a vector to deliver the CRISPR/cas9 system for treating genetic diseases and viral infections. However, the immunogenicity, toxicity, low transmission efficiency in brain and ear tissues, packaging size limitations of AAV, and immunogenicity and off-target effects of Cas9 protein pose several clinical challenges. This research reviews the role, challenges, and countermeasures of the AAV-CRISPR/cas9 system in gene therapy.

CRISPR (clustered regularly interspaced short palindromic repeats)/Cas gene editing is a revolutionary technology that can enable the correction of genetic mutations in vivo, providing great promise as a therapeutic intervention for inherited diseases. Adeno-associated viral (AAV) vectors are a potential vehicle for delivering CRISPR/Cas. However, they are restricted by their limited packaging capacity. Identifying smaller Cas orthologs that can be packaged, along with the required guide RNA elements, into a single AAV would be an important optimization for CRISPR/Cas gene editing. Expanding the options of Cas proteins that can be delivered by a single AAV not only increases translational application but also expands the genetic sites that can be targeted for editing. This review considers the benefits and current scope of small Cas protein orthologs that are suitable for gene editing approaches using single AAV vector delivery.

The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and its accompanying protein (Cas9) are now the most effective, efficient, and precise genome editing techniques. Two essential components of the CRISPR/Cas9 system are guide RNA (gRNA) and CRISPR-associated (Cas9) proteins. Choosing and implementing safe and effective delivery systems in the therapeutic application of CRISPR/Cas9 has proven to be a significant problem. For in vivo CRISPR/Cas9 delivery, viral vectors are the natural specialists. Due to their higher delivery effectiveness than other delivery methods, vectors such as adenoviral vectors (AdVs), adeno-associated viruses (AAVs), and lentivirus vectors (LVs) are now commonly employed as delivery methods. This review thoroughly examined recent achievements in using a variety of viral vectors as a means of CRISPR/Cas9 delivery, as well as the benefits and limitations of each viral vector. Future thoughts for overcoming the current restrictions and adapting the technology are also discussed.

… CRISPR-induced mutations in less than 3 h. Finally, we provide original evidence that AAV/CRISPR vectors … further improvement of AAV as CRISPR delivery tool. First, the efficiency of …

The frontiers of precision medicine have been revolutionized by the development of Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR)/Cas9 as an editing tool. CRISPR/Cas9 has been used to develop animal models, understand disease mechanisms, and validate treatment targets. In addition, it is regarded as an effective tool for genome surgery when combined with viral delivery vectors. In this article, we will explore the various viral mechanisms for delivering CRISPR/Cas9 into tissues and cells, as well as the benefits and drawbacks of each method. We will also review the history and recent development of CRISPR and viral vectors and discuss their applications as a powerful tool in furthering our exploration of disease mechanisms and therapies.

A vaccine for smallpox is no longer administered to the general public, and there is no proven, safe treatment specific to poxvirus infections, leaving people susceptible to infections by smallpox and other zoonotic Orthopoxviruses such as monkeypox. Using vaccinia virus (VACV) as a model organism for other Orthopoxviruses, CRISPR–Cas9 technology was used to target three essential genes that are conserved across the genus, including A17L, E3L, and I2L. Three individual single guide RNAs (sgRNAs) were designed per gene to facilitate redundancy in rendering the genes inactive, thereby reducing the reproduction of the virus. The efficacy of the CRISPR targets was tested by transfecting human embryonic kidney (HEK293) cells with plasmids encoding both SaCas9 and an individual sgRNA. This resulted in a reduction of VACV titer by up to 93.19% per target. Following the verification of CRISPR targets, safe and targeted delivery of the VACV CRISPR antivirals was tested using adeno-associated virus (AAV) as a packaging vector for both SaCas9 and sgRNA. Similarly, AAV delivery of the CRISPR antivirals resulted in a reduction of viral titer by up to 92.97% for an individual target. Overall, we have identified highly specific CRISPR targets that significantly reduce VACV titer as well as an appropriate vector for delivering these CRISPR antiviral components to host cells in vitro.

Adeno-associated viral (AAV) vectors packaging the CRISPR-Cas9 system (AAV-CRISPR) can efficiently modify disease-relevant genes in somatic tissues with high efficiency. AAV vectors are a preferred delivery vehicle for tissue-directed gene therapy because of their ability to achieve sustained expression from largely non-integrating episomal genomes. However, for genome editizng applications, permanent expression of non-human proteins such as the bacterially derived Cas9 nuclease is undesirable. Methods are needed to achieve efficient genome editing in vivo, with controlled transient expression of CRISPR-Cas9. Here, we report a self-deleting AAV-CRISPR system that introduces insertion and deletion mutations into AAV episomes. We demonstrate that this system dramatically reduces the level of Staphylococcus aureus Cas9 protein, often greater than 79%, while achieving high rates of on-target editing in the liver. Off-target mutagenesis was not observed for the self-deleting Cas9 guide RNA at any of the predicted potential off-target sites examined. This system is efficient and versatile, as demonstrated by robust knockdown of liver-expressed proteins in vivo. This self-deleting AAV-CRISPR system is an important proof of concept that will help enable translation of liver-directed genome editing in humans.

Self-complementary AAV-packaged CRISPR-Cas9 genome editing components rescue Duchenne muscular dystrophy. Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disease caused by mutations in the dystrophin gene (DMD). Previously, we applied CRISPR-Cas9–mediated “single-cut” genome editing to correct diverse genetic mutations in animal models of DMD. However, high doses of adeno-associated virus (AAV) are required for efficient in vivo genome editing, posing challenges for clinical application. In this study, we packaged Cas9 nuclease in single-stranded AAV (ssAAV) and CRISPR single guide RNAs in self-complementary AAV (scAAV) and delivered this dual AAV system into a mouse model of DMD. The dose of scAAV required for efficient genome editing were at least 20-fold lower than with ssAAV. Mice receiving systemic treatment showed restoration of dystrophin expression and improved muscle contractility. These findings show that the efficiency of CRISPR-Cas9–mediated genome editing can be substantially improved by using the scAAV system. This represents an important advancement toward therapeutic translation of genome editing for DMD.

… in vitro and in vivo delivery of CRISPRs. Here, we develop a CRISPR delivery platform using adeno-associated viruses (AAVs), because they are the preferred vectors for gene transfer …

… CRISPR–Cas9 delivery by adeno-associated virus (AAV) holds … to deploy CRISPR–Cas9 with AAVs, delivery vectors that are … tropism via local or systemic delivery 6 . We and others …

… -vector-based delivery systems as well as non-viral delivery systems have been used to achieve in vivo CRISPR delivery; … editing after delivery via split intein versus a single AAV vector …

Abstract Gene therapy has long held promise to correct a variety of human diseases and defects. Discovery of the Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR), the mechanism of the CRISPR-based prokaryotic adaptive immune system (CRISPR-associated system, Cas), and its repurposing into a potent gene editing tool has revolutionized the field of molecular biology and generated excitement for new and improved gene therapies. Additionally, the simplicity and flexibility of the CRISPR/Cas9 site-specific nuclease system has led to its widespread use in many biological research areas including development of model cell lines, discovering mechanisms of disease, identifying disease targets, development of transgene animals and plants, and transcriptional modulation. In this review, we present the brief history and basic mechanisms of the CRISPR/Cas9 system and its predecessors (ZFNs and TALENs), lessons learned from past human gene therapy efforts, and recent modifications of CRISPR/Cas9 to provide functions beyond gene editing. We introduce several factors that influence CRISPR/Cas9 efficacy which must be addressed before effective in vivo human gene therapy can be realized. The focus then turns to the most difficult barrier to potential in vivo use of CRISPR/Cas9, delivery. We detail the various cargos and delivery vehicles reported for CRISPR/Cas9, including physical delivery methods (e.g. microinjection; electroporation), viral delivery methods (e.g. adeno-associated virus (AAV); full-sized adenovirus and lentivirus), and non-viral delivery methods (e.g. liposomes; polyplexes; gold particles), and discuss their relative merits. We also examine several technologies that, while not currently reported for CRISPR/Cas9 delivery, appear to have promise in this field. The therapeutic potential of CRISPR/Cas9 is vast and will only increase as the technology and its delivery improves.

PURPOSE: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has recently been adapted to enable efficient editing of the mammalian genome, opening novel avenues for therapeutic intervention of inherited diseases. In seeking to disrupt yellow fluorescent protein (YFP) in a Thy1-YFP transgenic mouse, we assessed the feasibility of utilizing the adeno-associated virus 2 (AAV2) to deliver CRISPR/Cas for gene modification of retinal cells in vivo. METHODS: Single guide RNA (sgRNA) plasmids were designed to target YFP, and after in vitro validation, selected guides were cloned into a dual AAV system. One AAV2 construct was used to deliver Streptococcus pyogenes Cas9 (SpCas9), and the other delivered sgRNA against YFP or LacZ (control) in the presence of mCherry. Five weeks after intravitreal injection, retinal function was determined using electroretinography, and CRISPR/Cas-mediated gene modifications were quantified in retinal flat mounts. RESULTS: Adeno-associated virus 2-mediated in vivo delivery of SpCas9 with sgRNA targeting YFP significantly reduced the number of YFP fluorescent cells of the inner retina of our transgenic mouse model. Overall, we found an 84.0% (95% confidence interval [CI]: 81.8-86.9) reduction of YFP-positive cells in YFP-sgRNA-infected retinal cells compared to eyes treated with LacZ-sgRNA. Electroretinography profiling found no significant alteration in retinal function following AAV2-mediated delivery of CRISPR/Cas components compared to contralateral untreated eyes. CONCLUSIONS: Thy1-YFP transgenic mice were used as a rapid quantifiable means to assess the efficacy of CRISPR/Cas-based retinal gene modification in vivo. We demonstrate that genomic modification of cells in the adult retina can be readily achieved by viral-mediated delivery of CRISPR/Cas.

Adeno-associated virus-mediated (AAV-mediated) CRISPR editing is a revolutionary approach for treating inherited diseases. Sustained, often life-long mutation correction is required for treating these diseases. Unfortunately, this has never been demonstrated with AAV CRISPR therapy. We addressed this question in the mdx model of Duchenne muscular dystrophy (DMD). DMD is caused by dystrophin gene mutation. Dystrophin deficiency leads to ambulation loss and cardiomyopathy. We treated 6-week-old mice intravenously and evaluated disease rescue at 18 months. Surprisingly, nominal dystrophin was restored in skeletal muscle. Cardiac dystrophin was restored, but histology and hemodynamics were not improved. To determine the underlying mechanism, we evaluated components of the CRISPR-editing machinery. Intriguingly, we found disproportional guide RNA (gRNA) vector depletion. To test whether this is responsible for the poor outcome, we increased the gRNA vector dose and repeated the study. This strategy significantly increased dystrophin restoration and reduced fibrosis in all striated muscles at 18 months. Importantly, skeletal muscle function and cardiac hemodynamics were significantly enhanced. Interestingly, we did not see selective depletion of the gRNA vector after intramuscular injection. Our results suggest that gRNA vector loss is a unique barrier for systemic AAV CRISPR therapy. This can be circumvented by vector dose optimization.

… novel AAV capsids, and minimizing the immune response. This review discusses current challenges in delivering CRISPR-Cas nuclease-based genome editing therapies using AAV …

The clustered regularly interspaced short palindromic repeat (CRISPR) system, an emerging tool for genome editing, has garnered significant public interest for its potential in treating genetic diseases. Despite the rapid advancements in CRISPR technology, the progress in developing effective delivery strategies lags, impeding its clinical application. Extracellular nanovesicles (EVs), either in their endogenous forms or with engineered modifications, have emerged as a promising solution for CRISPR delivery. These EVs offer several advantages, including high biocompatibility, biological permeability, negligible immunogenicity, and straightforward production. Herein, we first summarize various types of functional EVs for CRISPR delivery, such as unmodified, modified, engineered virus-like particles (VLPs), and exosome-liposome hybrid vesicles, and examine their distinct intracellular pathways. Then, we outline the cutting-edge techniques for functionalizing extracellular vesicles, involving producer cell engineering, vesicle engineering, and virus-like particle engineering, emphasizing the diverse CRISPR delivery capabilities of these nanovesicles. Lastly, we address the current challenges and propose rational design strategies for their clinical translation, offering future perspectives on the development of functionalized EVs.

Abstract Clustered regularly interspaced palindromic repeats (CRISPR) is a gene editing tool with tremendous therapeutic potential. Recently, ribonucleoprotein (RNP) complex‐based CRISPR systems have gained momentum due to their reduction of off‐target editing. This has coincided with the emergence of extracellular vesicles (EVs) as a therapeutic delivery vehicle due to its low immunogenicity and high capacity for manipulation. EVs are cell‐derived membranous nanoparticles which mediate the intercellular transfer of molecular components. Current technologies achieve CRISPR RNP encapsulation into EVs through EVs biogenesis, thereby avoiding unnecessary physical, chemical or biological manipulations to the vesicles directly. Herein, we identify sixteen EVs‐based CRISPR RNP encapsulation strategies, each with distinct genetic features to encapsulate CRISPR RNP. According to the molecular mechanism facilitating the encapsulation process, there are six strategies of encapsulating Cas9 RNP into virus‐like particles based on genetic fusion, seven into EVs based on protein tethering, and three based on sgRNA‐coupled encapsulation. Additionally, the incorporation of a targeting moiety to the EVs membrane surface through EVs biogenesis confers tropism and increases delivery efficiency to specific cell types. The targeting moieties include viral envelope proteins, recombinant proteins containing a ligand peptide, single‐chain fragment variable (scFv) antibodies, and integrins. However, current strategies still have a number of limitations which prevent their use in clinical trials. Among those, the incorporation of viral proteins for encapsulation of Cas9 RNP have raised issues of biocompatibility due to host immune response. Future studies should focus on genetically engineering the EVs without viral proteins, enhancing EVs delivery specificity, and promoting EVs‐based homology directed repair. Nevertheless, the integration of CRISPR RNP encapsulation and tropism technologies will provide strategies for the EVs‐based delivery of CRISPR RNP in gene therapy and disease treatment.

The advent of genome editing has kindled the hope to cure previously uncurable, life-threatening genetic diseases. However, whether this promise can be ultimately fulfilled depends on how efficiently gene editing agents can be delivered to therapeutically relevant cells. Over time, viruses have evolved into sophisticated, versatile, and biocompatible nanomachines that can be engineered to shuttle payloads to specific cell types. Despite the advances in safety and selectivity, the long-term expression of gene editing agents sustained by viral vectors remains a cause for concern. Cell-derived vesicles (CDVs) are gaining traction as elegant alternatives. CDVs encompass extracellular vesicles (EVs), a diverse set of intrinsically biocompatible and low-immunogenic membranous nanoparticles, and virus-like particles (VLPs), bioparticles with virus-like scaffold and envelope structures, but devoid of genetic material. Both EVs and VLPs can efficiently deliver ribonucleoprotein cargo to the target cell cytoplasm, ensuring that the editing machinery is only transiently active in the cell and thereby increasing its safety. In this review, we explore the natural diversity of CDVs and their potential as delivery vectors for the clustered regularly interspaced short palindromic repeats (CRISPR) machinery. We illustrate different strategies for the optimization of CDV cargo loading and retargeting, highlighting the versatility and tunability of these vehicles. Nonetheless, the lack of robust and standardized protocols for CDV production, purification, and quality assessment still hinders their widespread adoption to further CRISPR-based therapies as advanced “living drugs.” We believe that a collective, multifaceted effort is urgently needed to address these critical issues and unlock the full potential of genome-editing technologies to yield safe, easy-to-manufacture, and pharmacologically well-defined therapies. Finally, we discuss the current clinical landscape of lung-directed gene therapies for cystic fibrosis and explore how CDVs could drive significant breakthroughs in in vivo gene editing for this disease.

Extracellular vesicles (EVs) have emerged as a promising platform for gene delivery owing to their natural properties and phenomenal functions, being able to circumvent the significant challenges associated with toxicity, problematic biocompatibility, and immunogenicity of the standard approaches. These features are of particularly interest for targeted delivery of the emerging clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems. However, the current efficiency of EV-meditated transport of CRISPR/Cas components remains insufficient due to numerous exogenous and endogenous barriers. Here, we comprehensively reviewed the current status of EV-based CRISPR/Cas delivery systems. In particular, we explored various strategies and methodologies available to potentially improve the loading capacity, safety, stability, targeting, and tracking for EV-based CRISPR/Cas system delivery. Additionally, we hypothesise the future avenues for the development of EV-based delivery systems that could pave the way for novel clinically valuable gene delivery approaches, and may potentially bridge the gap between gene editing technologies and the laboratory/clinical application of gene therapies. Graphical Abstract

Engineered virus-like particles (eVLPs) have emerged as a promising class of delivery systems for genome editing agents. By combining the efficient cellular entry of viral vectors with the safety advantages of nonviral platforms, eVLPs enable transient delivery of ribonucleoproteins such as Cas9, base editors, and prime editors. Successive design strategies, from rational engineering to directed optimization, have progressively improved particle assembly, cargo stability, and editing efficiency. In parallel, pseudotyping approaches have expanded the versatility of eVLPs for cell-type-specific targeting. Recent preclinical studies highlight their potential across diverse applications, ranging from monogenic disease models to complex disorders, and support their advancement toward clinical translation. This review summarizes the structural and production principles of eVLPs, surveys key advances in their development, and discusses therapeutic opportunities and future challenges for their application in genome editing.

Cell membrane-based biovesicles (BVs) are important candidate drug delivery vehicles and comprise extracellular vesicles, virus-like particles, and lentiviral vectors. Here, we introduce a non-enzymatic assembly of purified BVs, supercharged proteins, and plasmid DNA called pDNA-scBVs. This multicomponent vehicle results from the interaction of negative sugar moieties on BVs and supercharged proteins that contain positively charged amino acids on their surface to enhance their affinity for pDNA. pDNA-scBVs were demonstrated to mediate floxed reporter activation in culture by delivering a Cre transgene. We introduced pDNA-scBVs containing both a CRE-encoding plasmid and a BV-packaged floxed reporter into the brains of Ai9 mice. Successful delivery of both payloads by pDNA-scBVs was confirmed with reporter signal in the striatal brain region. Overall, we developed a more efficient method to load isolated BVs with cargo that functionally modified recipient cells. Augmenting the natural properties of BVs opens avenues for adoptive extracellular interventions using therapeutic loaded cargo. The development of positive supercharged protein loading of cell membrane-based biovesicles, including extracellular vesicles, virus-like particles, and lentiviral vectors, as a strategy to deliver exogenous plasmid DNA to target cells in vitro and in vivo is presented.

Rapid progress in gene editing based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) has revolutionized the study of gene and genome function and genetic disease correction. While numerous genetically modified cellular and animal models have been created to understand biological processes, the clinical application of CRISPR/Cas tools has been impeded by off-targeting and delivery problems. It is generally accepted that the delivery of CRISPR in the form of a ribonucleoprotein complex (RNP) substantially reduces the time of DNA exposure to the effector nuclease, minimizing off-target effects and facilitating clinical usage. This review focuses on CRISPR/Cas RNP delivery with retro/lentiviral particles and exosomes, whose parallel production by cells transfected with viral vectors is underestimated. We critically evaluate specific mechanisms of extracellular particle formation and loading with CRISPR/Cas for each system. Additionally, the details of Cas-nanoparticle entry and uncoating, previously unappreciated in the context of gene editing efficiency, are discussed. Based on existing knowledge about the consequences of intervention in retroviral assembly, entry, or exosome formation, we outline the potential problems with CRISPR/Cas delivery using extracellular nanoparticles and ways to address them.

… VLPs can serve as efficient carriers for RNP payloads. Such systems have been successfully applied to deliver a range of CRISPR-… FBS EV-depleted was used to produce VLPs and EV …

Despite the recognized potential of nanoparticles, only a few formulations have progressed to clinical trials, and an even smaller number have been approved by the regulatory authorities and marketed. Virus-like particles (VLPs) have emerged as promising alternatives to most explored nanoparticles because of the absence of viral genetic material, their incapacity to replicate, mimicry of viral structure, and tropism conservation. Furthermore, VLPs can be surface functionalized with small molecules to improve circulation half-life and target specificity. Through the functionalization and coating of VLPs, it is possible to optimize the response properties to a given stimulus, such as heat, pH, an alternating magnetic field, or even enzymes. Surface functionalization can also modulate other properties, such as biocompatibility, stability, and specificity, deeming VLPs as potential vaccine candidates or delivery systems. In this review, we address the different types of functionalization of VLPs, their importance, and their consequent biomedical applications.

Emerging molecular therapies introduce enzymatic activity into cells by delivering genes, transcripts, or proteins. Owing to their robust cell-entry capacity, virus-like particles (VLPs) represent a technology of choice in genome editing, where low doses of heterologous proteins and nucleic acids are essential. However, clinical translation of VLP vectors is hindered by inadequate purification methods. Current approaches, relying primarily on ultracentrifugation, suffer from inconsistent product quality and poor scalability. Here, we report the development of a broadly applicable purification strategy that improves the purity and therapeutic efficacy of genome-editing VLPs. Considering the characteristic properties of murine leukemia virus-derived engineered VLPs and HIV-derived engineered nucleocytosolic vehicles for loading of programmable editors, we developed a workflow that involves single-modal and multimodal chromatographic steps, effectively removing host cell proteins and cell-culture contaminants while improving VLP integrity and biological activity. Our purified VLPs displayed superior protein composition, consistency, and enhanced functional delivery compared to VLPs partially purified by conventional ultracentrifugation methods. Mass spectrometric analysis revealed a substantial decrease in contaminants, with VLP-specific proteins comprising >90% of the final product. In vivo studies confirmed improved therapeutic outcomes when chromatographically purified VLPs were used. Our scalable purification platform addresses critical manufacturing bottlenecks and constitutes a starting point for further development of VLP therapeutics, enabling robust production of pure VLPs for diverse applications such as genome editing, vaccine development, and other uses that require intracellular protein delivery.

CRISPR/Cas9-based genome editing has quickly emerged as a powerful breakthrough technology for use in diverse settings across biomedical research and therapeutic development. Recent efforts toward understanding gene modification methods in vitro have led to substantial improvements in ex vivo genome editing efficiency. Because disease targets for genomic correction are often localized in specific organs, realization of the full potential of genomic medicines will require delivery of CRISPR/Cas9 systems targeting specific tissues and cells directly in vivo. In this Perspective, we focus on progress toward in vivo delivery of CRISPR/Cas components. Viral and nonviral delivery systems are both promising for gene editing in diverse tissues via local injection and systemic injection. We describe the various viral vectors and synthetic nonviral materials used for in vivo gene editing and applications to research and therapeutic models, and summarize opportunities and progress to date for both methods. We also discuss challenges for viral delivery, including overcoming limited packaging capacity, immunogenicity associated with multiple dosing, and the potential for off-target effects, and nonviral delivery, including efforts to increase efficacy and to expand utility of nonviral carriers for use in extrahepatic tissues and cancer. Looking ahead, additional advances in the safety and efficiency of viral and nonviral delivery systems for tissue- and cell-type-specific gene editing will be required to enable broad clinical translation. We provide a summary of current delivery systems used for in vivo genome editing, organized with respect to route of administration, and highlight immediate opportunities for biomedical research and applications. Furthermore, we discuss current challenges for in vivo delivery of CRISPR/Cas9 systems to guide the development of future therapies.

CRISPR–Cas gene editing and messenger RNA-based protein replacement therapy hold tremendous potential to effectively treat disease-causing mutations with diverse cellular origin. However, it is currently impossible to rationally design nanoparticles that selectively target specific tissues. Here, we report a strategy termed selective organ targeting (SORT) wherein multiple classes of lipid nanoparticles are systematically engineered to exclusively edit extrahepatic tissues via addition of a supplemental SORT molecule. Lung-, spleen- and liver-targeted SORT lipid nanoparticles were designed to selectively edit therapeutically relevant cell types including epithelial cells, endothelial cells, B cells, T cells and hepatocytes. SORT is compatible with multiple gene editing techniques, including mRNA, Cas9 mRNA/single guide RNA and Cas9 ribonucleoprotein complexes, and is envisioned to aid the development of protein replacement and gene correction therapeutics in targeted tissues. The addition of selective organ targeting molecules to nanoparticles allows the specific targeting of extrahepatic tissues, enabling gene editing of distinct cell populations outside the liver.

… /exosome-mediated packaging of the nucleic acid/protein tools and the cell/tissue-targeted delivery to be a viable way towards the clinical translation of the CRISPR/Cas9 technology. …

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing system has been a major technological breakthrough that has brought revolutionary changes to genome editing for therapeutic and diagnostic purposes and precision medicine. With the advent of the CRISPR/Cas9 system, one of the critical limiting factors has been the safe and efficient delivery of this system to cells or tissues of interest. Several approaches have been investigated to find delivery systems that can attain tissue-targeted delivery, lowering the chances of off-target editing. While viral vectors have shown promise for in vitro, in vivo and ex vivo delivery of CRISPR/Cas9, their further clinical applications have been restricted due to shortcomings including limited cargo packaging capacity, difficulties with large-scale production, immunogenicity and insertional mutagenesis. Rapid progress in nonviral delivery vectors, including the use of lipid, polymer, peptides, and inorganic nanoparticle-based delivery systems, has established nonviral delivery approaches as a viable alternative to viral vectors. This review will introduce the molecular mechanisms of the CRISPR/Cas9 gene editing system, current strategies for delivering CRISPR/Cas9-based tools, an overview of strategies for overcoming off-target genome editing, and approaches for improving genome targeting and tissue targeting. We will also highlight current developments and recent clinical trials for the delivery of CRISPR/Cas9. Finally, future directions for overcoming the limitations and adaptation of this technology for clinical trials will be discussed. Graphical Abstract

The earliest example of in vivo expression of exogenous mRNA is by direct intramuscular injection in mice without the aid of a delivery vehicle. The current state of the art for therapeutic nucleic acid delivery is lipid nanoparticles (LNP), which are composed of cholesterol, a helper lipid, a PEGylated lipid and an ionizable amine-containing lipid. The liver is the primary organ of LNP accumulation following intravenous administration and is also observed to varying degrees following intramuscular and subcutaneous routes. Delivery of nucleic acid to hepatocytes by LNP has therapeutic potential, but there are many disease indications that would benefit from non-hepatic LNP tissue and cell population targeting, such as cancer, and neurological, cardiovascular and infectious diseases. This review will concentrate on the current efforts to develop the next generation of tissue-targeted LNP constructs for therapeutic nucleic acids.

Messenger RNA (mRNA) is being used as part of an emerging class of biotherapeutics with great promise for preventing and treating a wide range of diseases, as well as encoding programmable nucleases for genome editing. However, mRNA's low stability and immunogenicity, as well as the impermeability of the cell membrane to mRNA greatly limit mRNA's potential for therapeutic use. Lipid nanoparticles (LNPs) are currently one of the most extensively studied nanocarriers for mRNA delivery and have recently been clinically approved for developing mRNA‐based vaccines to prevent COVID‐19. In this review, we summarize the latest advances in designing ionizable lipids and formulating LNPs for intracellular and tissue‐targeted mRNA delivery. Furthermore, we discuss the progress of intracellular mRNA delivery for spatiotemporally controlled CRISPR/Cas9 genome editing by using LNPs. Finally, we provide a perspective on the future of LNP‐based mRNA delivery for CRISPR/Cas9 genome editing and the treatment of genetic disorders.

For genetic therapies to have their intended benefit, delivery systems must be designed which reach disease‐affected organs with high efficiency. To accomplish this, gene delivery systems must overcome multiple intra‐ and extracellular barriers to avoid rapid clearance from the body and/or significant accumulation in off‐target sites which can lead to undesired side effects (e.g., genotoxicity, immunogenicity). This requires an in‐depth knowledge of biomolecular and biophysical interactions at the nano–bio interface to engineer gene vectors which preferentially access specific organs such as the liver, spleen, and brain after systemic administration. In this review, we will discuss the strategies employed to engineer genetic therapies which selectively target organs of interest after systemic administration. We focus on three major classes of nucleic acid delivery systems including adeno‐associated viruses, lipid nanoparticles, and polymeric nanoparticles (PNPs) which are all being explored for tissue‐specific gene delivery. We will go on to describe how new, highly efficient adeno‐associated virus variants as well as engineered lipid and PNPs can be discovered or rationally designed. We also discuss high throughput approaches for screening of these systems to establish important structure‐to‐function relationships that determine the fate of these gene delivery systems once administered.

Obesity is an increasing pathophysiological problem in developed societies. Despite all major progress in understanding molecular mechanisms of obesity, currently available anti-obesity drugs have shown limited efficacy with severe side effects. CRISPR interference (CRISPRi) mechanism based on catalytically dead Cas9 (dCas9) and single guide RNA (sgRNA) was combined with a targeted nonviral gene delivery system to treat obesity and obesity-induced type 2 diabetes. A fusion peptide targeting a vascular and cellular marker of adipose tissue, prohibitin, was developed by conjugation of adipocyte targeting sequence (CKGGRAKDC) to 9-mer arginine (ATS-9R). (dCas9/sgFabp4) + ATS-9R oligoplexes showed effective condensation and selective delivery into mature adipocytes. Targeted delivery of the CRISPRi system against Fabp4 to white adipocytes by ATS-9R induced effective silencing of Fabp4, resulting in reduction of body weight and inflammation and restoration of hepatic steatosis in obese mice. This RNA-guided DNA recognition platform provides a simple and safe approach to regress and treat obesity and obesity-induced metabolic syndromes.

With the advent of new genome editing technologies and the emphasis placed on their optimization, the genetic and phenotypic correction of a plethora of diseases sit on the horizon. Ideally, genome editing approaches would provide long-term solutions through permanent disease correction instead of simply treating patients symptomatically. Although various editing machinery options exist, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated protein) editing technique has emerged as the most popular due to its high editing efficiency, simplicity, and affordability. However, while CRISPR technology is gradually being perfected, optimization is futile without accessible, effective, and safe delivery to the desired cell or tissue. Therefore, it is important that scientists simultaneously focus on inventing and improving delivery modalities for editing machinery as well. In this review, we will discuss the critical details of viral and nonviral delivery systems, including payload, immunogenicity, efficacy in delivery, clinical application, and future directions.

The capacity to efficiently deliver the gene-editing enzyme complex to target cells is favored over other forms of gene delivery as it offers one-time hit-and-run gene editing, thus improving precision and safety and reducing potential immunogenicity against edited cells in clinical applications. Here we performed a proof-of-mechanism study and demonstrated that a simian adenoviral vector for DNA delivery can be repurposed as a robust intracellular delivery platform for a functional Cas9/guide RNA (gRNA) complex to recipient cells. In this system, the clinically relevant adenovirus was genetically engineered with a plug-and-display technology based on SpyTag003/SpyCatcher003 coupling chemistry. Under physiological conditions, an off-the-shelf mixture of viral vector with SpyTag003 incorporated into surface capsid proteins and Cas9 fused with SpyCatcher003 led to a rapid titration reaction yielding adenovirus carrying Cas9SpyCatcher003 on the virus surface. The Cas9 fusion protein-conjugated viruses in the presence of a reporter gRNA delivered gene-editing functions to cells with an efficiency comparable to that of a commercial CRISPR/Cas9 transfection reagent. Our data fully validate the adenoviral “piggyback” approach to deliver an intracellularly acting enzyme cargo and, thus, warrant the prospect of engineering tissue-targeted adenovirus carrying Cas9/gRNA for in vivo gene editing.

… nanocarriers enabling precise delivery and protection of the CRISPR-Cas9 system (Figure 5A). Casgevy’s clinical trial data provide direct evidence for the translational value of LNP …

Advancements in antiretroviral therapy (ART) enable those living with the human immunodeficiency virus type one (HIV‐1) to lead longer, healthier lives free from disease comorbidities. However, lifelong ART poses challenges. These include social stigma, medication costs, drug accessibility, mental health, and drug‐related toxicities. Moreover, ART does not eliminate latent HIV‐1 DNA. Viral persistence in tissue and cell reservoirs results in viral rebound after ART interruption. New strategies are required to achieve a functional HIV‐1 cure. To excise latent HIV‐1, C‐X‐C motif chemokine receptor 4 (CXCR4) ligand‐decorated lymphoid tissue‐targeting lipid nanoparticles (LNPs) for CRISPR‐Cas9/gRNA delivery are developed. These LNPs enhance mRNA translation and demonstrate CXCR4‐mediated improved uptake to eliminate HIV‐1 DNA in infected CD4+ T cells. LNPs also facilitate targeted drug delivery, achieving HIV‐1 DNA excision in ART‐treated, infected humanized mice. This study emphasizes the potential of tissue and cell‐targeted LNPs for effective HIV‐1 DNA excision.

… CRISPR–Cas9 genome editing in a therapeutic mouse model through the delivery of mRNA … These top-performing tissue-targeted SiLNPs adsorb various proteins on their surface, …

Genome editing holds great potential for cancer treatment due to the ability to precisely inactivate or repair cancer-related genes. However, delivery of CRISPR/Cas to solid tumours for efficient cancer therapy remains challenging. Here we targeted tumour tissue mechanics via a multiplexed dendrimer lipid nanoparticle (LNP) approach involving co-delivery of focal adhesion kinase (FAK) siRNA, Cas9 mRNA and sgRNA (siFAK + CRISPR-LNPs) to enable tumour delivery and enhance gene-editing efficacy. We show that gene editing was enhanced >10-fold in tumour spheroids due to increased cellular uptake and tumour penetration of nanoparticles mediated by FAK-knockdown. siFAK + CRISPR-PD-L1-LNPs reduced extracellular matrix stiffness and efficiently disrupted PD-L1 expression by CRISPR/Cas gene editing, which significantly inhibited tumour growth and metastasis in four mouse models of cancer. Overall, we provide evidence that modulating the stiffness of tumour tissue can enhance gene editing in tumours, which offers a new strategy for synergistic LNPs and other nanoparticle systems to treat cancer using gene editing. In vivo delivery of the CRISPR/Cas system is a promising cancer therapy approach, but its efficacy is hampered by low penetrability of nanoparticles in the stiff tumour tissue. Here the authors use dendrimer lipid nanoparticles to couple PD-L1 gene editing with knockdown of FAK, a protein involved in cell adhesion, showing that modulation of the mechanical properties of tumour cells leads to enhanced gene editing and tumour growth inhibition in four different animal models.

… -based delivery systems have recently … delivery of CRISPR-Cas 9. In this chapter, we summarize the recent advancements in CRISPR-Cas9-delivering nanocarrier-based drug delivery …