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Gene therapy for sickle cell disease: recent advances, clinical trials and future directions.
Sickle cell disease (SCD) is the most common inherited blood disorder worldwide, impacting millions and imposing severe healthcare challenges, particularly in resource-limited regions. Current treatments have variable efficacy and require lifelong adherence. Allogeneic Hematopoietic Stem Cell Transplantation can be curative but comes with significant side effects and limited donor availability limits its widespread applicability. Gene therapy, by addressing the root genetic causes, offers a revolutionary alternative. This article discusses the molecular mechanisms of SCD and β-thalassemia and highlights advancements in gene therapy, such as gene addition via lentiviral vectors and gene editing with CRISPR/Cas9 technology. Clinical trials have brought about significant progress but challenges remain, including leukemogenesis, delivery efficiency and cost. Future efforts must focus on enhancing efficiency, reducing costs, developing nongenotoxic conditioning regimens and methods for in vivo application.
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Stimuli-responsive peptide nanocarriers for tumor-specific CRISPR/Cas9 delivery and precision genome editing.
CRISPR/Cas9 ribonucleoprotein (RNP) delivery remains a critical challenge due to its large size, instability, and off-target effects. Here, we report a stimuli-responsive cationic amphiphilic peptide, (CR3)3C, designed for cancer-targeted delivery of CRISPR/Cas9 RNP. The peptide integrates three functional domains: (1) a naphthyl-diphenylalanine (Nap-FF) motif enabling self-assembly into stable nanoparticles via aromatic interactions, (2) a matrix metalloproteinase-7 (MMP7)-cleavable linker (GPLGLA) for tumor microenvironment-specific activation, and (3) a redox-responsive cationic domain ((CRRR)3-C) for electrostatic RNP binding and glutathione (GSH)-triggered intracellular release. The (CR3)3C/RNP nanocomplexes (108.8 nm diameter, ζ = +10.89 mV) demonstrate exceptional stability and cellular uptake efficiency. Mechanistic studies reveal caveolae-mediated endocytosis and lipid raft-associated pathways, proton sponge effect-driven endosomal escape, and nuclear localization facilitated by Cas9's nuclear localization signal. In HeLa-EGFP cells, (CR3)3C/RNP shows 33.8 % gene editing efficiency at 100 nM RNP with >90 % cell viability. This work establishes a programmable, non-viral platform that synergizes enzymatic and redox responsiveness for tumor-targeted genome editing, addressing critical barriers in CRISPR therapeutics.
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Engineering a compact genome-editing tool.
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Delivering genome editing tools to primary cells.
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Human germline editing needs one message.
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Graphene oxide-mediated Cas9/sgRNA delivery for efficient genome editing.
Direct cellular delivery of CRISPR/Cas9 complexes is of great significance for genome editing and other recently developed applications, such as gene expression regulation and RNA/DNA imaging. Here, we first constructed a graphene oxide (GO)-polyethylene glycol (PEG)-polyethylenimine (PEI) nanocarrier for the delivery of high-molecular-weight Cas9/single-guide RNA (sgRNA) complexes for endocytosis, endosomal escape, nuclear entry, and gene editing. The results demonstrate that the nanocarrier can be used successfully for efficient gene editing in human AGS cells with an efficiency of ∼39%. The results also show that this nanocarrier can protect sgRNA from enzymatic degradation, thus exhibiting extremely high stability, which is critical for future in vivo applications. Thus, this GO-mediated Cas9/sgRNA delivery system has potential as a new approach for biomedical research and targeted gene engineering applications.
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CRISPR still needs microbiologists.
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Creation of zebrafish knock-in reporter lines in the nefma gene by Cas9-mediated homologous recombination.
CRISPR/Cas9-based strategies are widely used for genome editing in many organisms, including zebrafish. Although most applications consist in introducing double strand break (DSB)-induced mutations, it is also possible to use CRISPR/Cas9 to enhance homology directed repair (HDR) at a chosen genomic location to create knock-ins with optimally controlled precision. Here, we describe the use of CRISPR/Cas9-targeted DSB followed by HDR to generate zebrafish transgenic lines where exogenous coding sequences are added in the nefma gene, in frame with the endogenous coding sequence. The resulting knock-in embryos express the added gene (fluorescent reporter or KalTA4 transactivator) specifically in the populations of neurons that express nefma, making them convenient tools for research on these populations.
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Lipopolymer Nanoparticle-Mediated In Vivo CRISPR-Cas9 Editing of Mrg15 Restores Mitochondrial Mitophagy to Alleviate Metabolic-Associated Steatohepatitis.
Metabolic-associated steatohepatitis (MASH) remains difficult to treat due to the lack of interventions capable of targeting upstream disease drivers and achieving durable disease modification. The epigenetic regulator Mrg15 has been implicated in mitochondrial dysfunction and metabolic stress in the liver, suggesting its potential relevance to MASH pathogenesis. Here, we develop a liver-targeted lipopolymer nanoparticle (LPNP)-mediated gene editing platform to enable in vivo disruption of Mrg15 by co-delivery of Cas9 mRNA and Mrg15 sgRNA. The screened P64H/Mrg15 system achieved efficient hepatic delivery and genome editing, resulting in reduced Mrg15 expression in hepatocytes. In the MASH mouse model, P64H/Mrg15 treatment was associated with decreased hepatic lipid accumulation, improved liver injury markers, and attenuation of inflammation and fibrosis. Sequence-level analyses confirmed on-target editing in liver tissue, and systemic histopathological evaluation revealed no overt toxicity in major organs under the tested dosing regimen. Transcriptomic profiling revealed coordinated pathway-level associations involving metabolic, inflammatory, and autophagy-related regulation, while protein-level analyses demonstrated alterations in selected autophagy- and mitophagy-related regulators, including TUFM and LC3B-II. Together, these findings identify Mrg15 as a disease-relevant epigenetic regulator in MASH and highlight liver-directed P64H LPNP/CRISPR delivery as a promising non-viral strategy for modulating upstream regulatory pathways in metabolic liver disease.
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Gene targeting in adult organs using in vivo cleavable donor plasmids for CRISPR-Cas9 and CRISPR-Cas12a.
The CRISPR-Cas system for in vivo genome editing is a powerful tool for gene therapy against several diseases. We have previously developed the pCriMGET_9-12a system, an in vivo cleavable donor plasmid for precise targeted knock-in of exogenous DNA by both Cas9 and Cas12a. Here, we show that the pCriMGET_9-12a system can be applied for in vivo in-frame knock-in of exogenous DNA in adult mouse liver by hydrodynamic delivery of the targeting plasmids. The in vivo cleavable pCriMGET_9-12a donor plasmids significantly increased the knock-in efficiency of both CRISPR-Cas9 and CRISPR-Cas12a in the adult mouse liver compared to uncleavable donor plasmids. This strategy also achieved in-frame reporter gene knock-in without indel mutations. Therefore, in vivo gene targeting using the pCriMGET_9-12a system may contribute to the establishment of safer, more precise, versatile and efficient gene therapy methods in adult organs.
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