Cited by <i>In vivo</i>Treatment of a Severe Vascular Disease via a Bespoke CRISPR-Cas9 Base Editor

mTORopathies in Epilepsy and Neurodevelopmental Disorders: The Future of Therapeutics and the Role of Gene Editing DOI

Marina Ottmann Boff, Fernando Antônio Costa Xavier, Fernando Mendonça Diz

et al.

Cells, Journal Year: 2025, Volume and Issue: 14(9), P. 662 - 662

Published: April 30, 2025

mTORopathies represent a group of neurodevelopmental disorders linked to dysregulated mTOR signaling, resulting in conditions such as tuberous sclerosis complex, focal cortical dysplasia, hemimegalencephaly, and Smith–Kingsmore Syndrome. These often manifest with epilepsy, cognitive impairments, and, some cases, structural brain anomalies. The pathway, central regulator cell growth metabolism, plays crucial role development, where its hyperactivation leads abnormal neuroplasticity, tumor formation, heightened neuronal excitability. Current treatments primarily rely on inhibitors, rapamycin, which reduce seizure frequency size but fail address underlying genetic causes. Advances gene editing, particularly via CRISPR/Cas9, offer promising avenues for precision therapies targeting the mutations driving mTORopathies. New delivery systems, including viral non-viral vectors, aim enhance specificity efficacy these therapies, potentially transforming management disorders. While editing holds curative potential, challenges remain concerning delivery, long-term safety, ethical considerations. Continued research into mechanisms innovative may pave way transformative, personalized patients affected by complex conditions.

Language: Английский

Citations

Lipo-Xenopeptide Polyplexes for CRISPR/Cas9 based Gene editing at ultra-low dose DOI

Janin Germer,

Anna-Lina Lessl,

Jana Pöhmerer

et al.

Journal of Controlled Release, Journal Year: 2024, Volume and Issue: 370, P. 239 - 255

Published: April 27, 2024

Double pH-responsive xenopeptide carriers containing succinoyl tetraethylene pentamine (Stp) and lipo amino fatty acids (LAFs) were evaluated for CRISPR/Cas9 based genome editing. Different carrier topologies, variation of LAF/Stp ratios LAF types as Cas9 mRNA/sgRNA polyplexes screened in three different reporter cell lines using genomic targets (Pcsk9, eGFP, mdx exon 23). One U-shaped bundle (B2)-shaped lipo-xenopeptides exhibiting remarkable efficiencies identified. Genome editing potency top observed at sub-nanomolar EC

Language: Английский

Citations

Dual pH-responsive CRISPR/Cas9 ribonucleoprotein xenopeptide complexes for genome editing DOI

Xianjin Luo,

Janin Germer,

Tobias Burghardt

et al.

European Journal of Pharmaceutical Sciences, Journal Year: 2024, Volume and Issue: 205, P. 106983 - 106983

Published: Dec. 7, 2024

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated (Cas) protein has been proved as a powerful tool for the treatment of genetic diseases. The Cas9 protein, when combined with single-guide RNA (sgRNA), forms Cas9/sgRNA ribonucleoprotein (RNP) capable targeting and editing genome. However, limited availability effective carriers restricted broader application CRISPR/Cas9 RNP. In this study, we evaluated dual pH-responsive amphiphilic xenopeptides (XPs) delivering These artificial lipo-XPs contain apolar cationizable lipoamino fatty acid (LAF) polar oligoaminoethylene units such succinoyl-tetraethylenepentamine (Stp) in various ratios U-shaped topologies. were screened functional RNP delivery four different reporter cell lines, including Duchenne muscular dystrophy (DMD) exon skipping model. Significantly enhanced cellular uptake into HeLa cells, endosomal disruption gal8-mRuby3 potent genome by several complexes was observed lines 5 nM sgRNA range. Comparing mRNA/sgRNA polyplexes DMD model demonstrated similar splice site high two molecular modalities. Based on these studies, analogues U1 LAF2-Stp LAF4-Stp2 structures deployed, tuning amphiphilicity Stp group replacement six oligoamino acids dmGtp, chGtp, dGtp, Htp, Stt, or GEIPA. most (containing chGtp GEIPA) further gene efficiency EC50 values 1 line. Notably, LAF2-dGtp reached 0.51 even upon serum incubation. Another carrier (LAF4-GEIPA2) complexing donor DNA, facilitated up to 43 % homology-directed repair (HDR) eGFPd2 cells visualized switch from green fluorescent (eGFP) blue (BFP). This study presents system tunable RNP/donor DNA polyplexes, offering an easily applicable strategy editing.

Language: Английский

Citations

Development of an RNA virus-based episomal vector with artificial aptazyme for gene silencing DOI

Ryo Komorizono,

Shima Yoshizumi,

Keizō Tomonaga

et al.

Applied Microbiology and Biotechnology, Journal Year: 2024, Volume and Issue: 108(1)

Published: Oct. 18, 2024

Abstract RNA virus-based episomal vector (REVec), engineered from Borna disease virus, is an innovative gene delivery tool that enables sustained expression in transduced cells. However, the difficulty controlling and eliminating vectors has limited practical use of REVec. In this study, we overcome these shortcomings by inserting artificial aptazymes into untranslated regions foreign genes carried or downstream viral phosphoprotein gene, which essential for replication. Non-transmissive REVec carrying GuaM8HDV P1-F5 aptazyme showed immediate suppression a guanine theophylline concentration-dependent manner. Continuous compound administration also markedly reduced percentage vector-transduced cells eventually led to complete elimination This new safe technology allows fine-tuning could be useful platform therapy gene-cell therapy, potentially contributing cure many genetic disorders. Key points • We developed bornavirus capable silencing transgene insertion Transgene was suppressed manner Artificial systems allowed

Language: Английский

Citations

Non-viral gene therapy for Leber’s congenital amaurosis: progress and possibilities DOI

Latifat Abdulsalam,

James Mordecai,

Irshad Ahmad

et al.

Nanomedicine, Journal Year: 2024, Volume and Issue: unknown, P. 1 - 14

Published: Dec. 20, 2024

Leber's congenital amaurosis (LCA) represents a set of rare and pervasive hereditary conditions the retina that cause severe vision loss starting in early childhood. Targeted treatment intervention has become possible thanks to recent advances understanding LCA genetic basis. While viral vectors have shown efficacy gene delivery, they present challenges related safety, low cargo capacity, potential for random genomic integration. Non-viral therapy is safer more flexible alternative treating underlying mutation causing LCA. delivery methods, such as inorganic nanoparticles, polymer-based systems, lipid-based bypass risks immunogenicity integration, potentially offering versatile personalized patients. This review explores background LCA, emphasizing mutations involved, diverse non-viral methods being developed. It also highlights studies on animal models clinical trials. presents future perspectives therapy, including integrating emerging technologies like CRISPR-Cas9, interdisciplinary collaborations, medicine, ethical considerations.

Language: Английский

Citations

Current and Emerging Issues in Adeno-Associated Virus Vector-Mediated Liver-Directed Gene Therapy DOI

Pasquale Piccolo, Nicola Brunetti‐Pierri

Human Gene Therapy, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 23, 2024

Adeno-associated virus (AAV) vectors have demonstrated safety and efficacy for gene transfer to hepatocytes in preclinical models, various clinical trials from a experience with growing number of approved therapy products. Although the exact duration is unknown, expression therapeutic genes remains stable several years after single administration vector at clinically relevant doses adult patients hemophilia other inherited metabolic disorders. However, applications, especially diseases requiring high AAV by intravenous administrations, raised concerns. These include prevalence pre-existing immunity against capsid, activation complement innate serious life-threatening complications, elevation liver transaminases, growth associated loss transgene expression, underlying conditions negatively affecting efficacy. Despite these issues, field rapidly advancing better understanding vector-host interactions development new strategies improve liver-directed therapy. This review provides an overview current emerging challenges AAV-mediated

Language: Английский

Citations

Modulating efficacy and cytotoxicity of lipoamino fatty acid nucleic acid carriers using disulfide or hydrophobic spacers DOI

Ricarda C. Steffens,

Sophie Thalmayr,

Eric Weidinger

et al.

Nanoscale, Journal Year: 2024, Volume and Issue: 16(29), P. 13988 - 14005

Published: Jan. 1, 2024

Double pH-responsive xenopeptides comprising polar ionizable succinoyl tetraethylene pentamine (Stp) motifs and lipophilic lipoamino fatty acids (LAFs) were recently found to efficiently transfect mRNA pDNA at low doses.

Language: Английский

Citations

Ex Vivo Machine Perfusion as a Platform for Lentiviral Gene Delivery in Rat Livers DOI

Korkut Uygun, Irina Filz von Reiterdank,

Mohammadreza Mojoudi

et al.

Research Square (Research Square), Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 13, 2024

Developing new strategies for local monitoring and delivery of immunosuppression is critical to making allografts safer more accessible. Ex vivo genetic modification grafts using machine perfusion presents a promising approach improve graft function modulate immune responses while minimizing risks off-target effects systemic immunogenicity in vivo. This proof-of-concept study demonstrates the feasibility normothermic (NMP) mimic vitro conditions effective gene delivery. In this study, lentiviral vectors carrying biosensor constructs with Gaussia Luciferase (GLuc) were introduced rodent livers during 72-hour period, targeted 3 x 10

Language: Английский

Citations

Use of paired Cas9-NG nickase and truncated sgRNAs for single-nucleotide microbial genome editing DOI

Song Hee Jeong, Ho Joung Lee, Sang Jun Lee

et al.

Frontiers in Genome Editing, Journal Year: 2024, Volume and Issue: 6

Published: Sept. 26, 2024

The paired nickases approach, which utilizes clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) nickase and dual guide RNA, has the advantage of reducing off-target effects by being able to double target sequence. In this study, our research utilized Cas9-NG variant minimize PAM sequence constraints, enabling generation nicks at desired genomic loci. We performed a systematic investigation into formation sites for design donor DNA within bacterial model system. Although we successfully identified conditions necessary effective in vivo , achieving single-nucleotide level editing directly genome proved challenging. Nonetheless, experiments revealed that efficient was achievable on sequences are hybridized with 5′-end-truncated single-guide RNAs (sgRNAs). Our findings contribute deeper understanding offering single-mismatch intolerance strategy accurate nucleotide editing. This not only enhances precision but also marks significant step forward development nickase-derived technologies.

Language: Английский

Citations

In vivoTreatment of a Severe Vascular Disease via a Bespoke CRISPR-Cas9 Base Editor DOI

Christiano R. R. Alves, Sabyasachi Das,

Vijai Krishnan

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 11, 2024

Genetic vascular disorders are prevalent diseases that have diverse etiologies and few treatment options. Pathogenic missense mutations in the alpha actin isotype 2 gene (

Language: Английский

Citations