Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs

Molecular Therapy, Journal Year: 2023, Volume and Issue: 31(8), P. 2422 - 2438

Published: July 4, 2023

Transient delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) into the central nervous system (CNS) for therapeutic genome editing could avoid limitations viral vector-based including cargo capacity, immunogenicity, and cost. Here, we tested ability cell-penetrant Cas9 RNPs to edit mouse striatum when introduced using a convection-enhanced system. These transient showed comparable neurons reduced adaptive immune responses relative one formulation delivered AAV serotype 9. The production ultra-low endotoxin protein manufactured at scale further improved innate immunity. We conclude that injection-based minimally immunogenic CRISPR CNS provides valuable alternative virus-mediated editing.

Language: Английский

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Transient inhibition of 53BP1 increases the frequency of targeted integration in human hematopoietic stem and progenitor cells

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: Jan. 2, 2024

Abstract Genome editing by homology directed repair (HDR) is leveraged to precisely modify the genome of therapeutically relevant hematopoietic stem and progenitor cells (HSPCs). Here, we present a new approach increasing frequency HDR in human HSPCs delivery an inhibitor 53BP1 (named “i53”) as recombinant peptide. We show that use i53 peptide effectively increases HDR-mediated at variety loci well other primary cell types. incorporating protein allows high frequencies while lowering amounts AAV6 needed 8-fold. edited were capable long-term bi-lineage reconstitution NSG mice, suggesting might be safely integrated into standard CRISPR/AAV6-mediated protocol gain greater numbers for transplantation clinically meaningful populations.

Language: Английский

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Exonic knockout and knockin gene editing in hematopoietic stem and progenitor cells rescues RAG1 immunodeficiency

Science Translational Medicine, Journal Year: 2024, Volume and Issue: 16(733)

Published: Feb. 7, 2024

Recombination activating genes ( RAGs ) are tightly regulated during lymphoid differentiation, and their mutations cause a spectrum of severe immunological disorders. Hematopoietic stem progenitor cell (HSPC) transplantation is the treatment choice but limited by donor availability toxicity. To overcome these issues, we developed gene editing strategies targeting corrective sequence into human RAG1 homology-directed repair (HDR) validated them tailored two-dimensional, three-dimensional, in vivo xenotransplant platforms to assess rescue expression function. Whereas integration intron 1 achieved suboptimal correction, in-frame insertion exon 2 drove physiologic activity, allowing disruption dominant-negative effects unrepaired hypomorphic alleles. Enhanced HDR-mediated enabled correction HSPCs from patients with T B differentiation blocks. Gene efficiency exceeded minimal proportion functional required immunodeficiency Rag1 –/– mice, supporting clinical translation HSPC for deficiency.

Language: Английский

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Gene therapy for β-thalassemia: current and future options

Trends in Molecular Medicine, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 1, 2025

Language: Английский

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The p53 challenge of hematopoietic stem cell gene editing

Molecular Therapy — Methods & Clinical Development, Journal Year: 2023, Volume and Issue: 30, P. 83 - 89

Published: June 12, 2023

gene editing in hematopoietic stem and progenitor cells (HSPCs) represents a promising curative treatment strategy for monogenic blood disorders. Gene using the homology-directed repair (HDR) pathway enables precise genetic modifications ranging from single base pair correction to replacement or insertion of large DNA segments. Hence, HDR-based could facilitate broad application across disorders, but technology still faces challenges clinical translation. Among these, recent studies demonstrate induction damage response (DDR) p53 activation caused by double-strand breaks exposure recombinant adeno-associated virus vector templates, resulting reduced proliferation, engraftment, clonogenic capacity edited HSPCs. While different mitigation strategies can reduce this DDR, more research is needed on phenomenon ensure safe efficient implementation clinic.

Language: Английский

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Enrichment strategies to enhance genome editing

Journal of Biomedical Science, Journal Year: 2023, Volume and Issue: 30(1)

Published: July 1, 2023

Abstract Genome editing technologies hold great promise for numerous applications including the understanding of cellular and disease mechanisms development gene therapies. Achieving high frequencies is critical to these research areas achieve overall goal being able manipulate any target with desired genetic outcome. However, sometimes suffer from low efficiencies due several challenges. This often case emerging technologies, which require assistance translation into broader applications. Enrichment strategies can support this by selecting edited cells non-edited cells. In review, we elucidate different enrichment strategies, their many in non-clinical clinical settings, remaining need novel further improve genome therapy studies.

Language: Английский

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The recombinase activating genes: architects of immune diversity during lymphocyte development

Frontiers in Immunology, Journal Year: 2023, Volume and Issue: 14

Published: July 11, 2023

The mature lymphocyte population of a healthy individual has the remarkable ability to recognise an immense variety antigens. Instead encoding unique gene for each potential antigen receptor, evolution used rearrangements, also known as variable, diversity, and joining segment (V(D)J) recombination. This process is critical development relies on recombination-activating genes-1 (RAG1) RAG2, here collectively referred RAG. RAG serves powerful genome editing tools lymphocytes strictly regulated prevent dysregulation. However, in case dysregulation, been implicated cases cancer, autoimmunity severe combined immunodeficiency (SCID). review examines functional protein domains motifs RAG, describes advances our understanding function (dys)regulation discuss new therapeutic options, such therapy, deficiencies, explore

Language: Английский

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Sickle Cell Disease: From Genetics to Curative Approaches

Annual Review of Genomics and Human Genetics, Journal Year: 2023, Volume and Issue: 24(1), P. 255 - 275

Published: Aug. 25, 2023

Sickle cell disease (SCD) is a monogenic blood caused by point mutation in the gene coding for β-globin. The abnormal hemoglobin [sickle (HbS)] polymerizes under low-oxygen conditions and causes red cells to sickle. clinical presentation varies from very severe (with acute pain, chronic early mortality) normal (few complications life span). variability of SCD might be due (in part) various genetic modulators. First, we review main factors, polymorphisms, modifier genes that influence expression globin or otherwise modulate severity SCD. Considering as complex, multifactorial disorder important development appropriate pharmacological treatments. Second, characteristics, advantages, disadvantages latest advances therapy SCD, lentiviral-vector-based approaches gene-editing strategies.

Language: Английский

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Understanding and Tackling Immune Responses to Adeno-Associated Viral Vectors

Human Gene Therapy, Journal Year: 2023, Volume and Issue: 34(17-18), P. 836 - 852

Published: Sept. 1, 2023

As the clinical experience in adeno-associated viral (AAV) vector-based gene therapies is expanding, necessity to better understand and control host immune responses also increasing. Immunogenicity of AAV vectors humans has been linked several limitations platform, including lack efficacy due antibody-mediated neutralization, tissue inflammation, loss transgene expression, some cases, complement activation acute toxicities. Nevertheless, significant knowledge gaps remain our understanding mechanisms therapies, further hampered by failure preclinical animal models recapitulate findings. In this review, we focus on current regarding responses, spanning from innate immunity humoral adaptive triggered how they can be mitigated for safer, durable, more effective therapies.

Language: Английский

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Advances in gene therapy for inborn errors of immunity

Current Opinion in Allergy and Clinical Immunology, Journal Year: 2023, Volume and Issue: 23(6), P. 467 - 477

Published: Oct. 13, 2023

Purpose of review Provide an overview the landmark accomplishments and state art gene therapy for inborn errors immunity (IEI). Recent findings Three decades after first clinical application IEI, there is one market authorized product available, while several others efficacy has been demonstrated or currently being tested in ongoing trials. Gene editing approaches using programmable nucleases are explored preclinically could be beneficial genes requiring tightly regulated expression, gain-of-function mutations dominant-negative mutations. Summary by modifying autologous hematopoietic stem cells (HSCs) offers attractive alternative to allogeneic cell transplantation (HSCT), current standard care treat severe IEI. This approach does not require availability a suitable donor eliminates risk graft versus host disease (GvHD). can attempted viral vector add copy therapeutic (viral addition) (gene editing) precisely correct mutations, disrupt introduce entire at specific locus. However, comes with its own challenges such as safety, effectiveness access. For addition, major safety concern vector-related insertional mutagenesis, although this greatly reduced introduction safer vectors. editing, off-site mutagenesis main driver behind search modified nucleases. both approaches, HSCs have manipulated ex vivo, doing efficiently without losing stemness remains challenge, especially editing.

Language: Английский

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