Genetic engineering meets hematopoietic stem cell biology for next-generation gene therapy

Ferrari, Samuele; Valeri, Erika; Conti, Anastasia; Scala, Serena; Aprile, Annamaria; Micco, Raffaella Di; Kajaste‐Rudnitski, Anna; Montini, Eugenio; Ferrari, Giuliana; Aiuti, Alessandro; Naldini, Luigi

doi:10.1016/j.stem.2023.04.014

Cited by 30 publications

(9 citation statements)

References 267 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Gene editing represents a promising tool to engineer human hematopoietic stem/progenitor cells (HSPCs), opening the possibility to precisely correct disease-causing mutations while limiting the risk of genome-wide insertional mutagenesis and unregulated expression of the transgene 1 . In the last decade, CRISPR-Cas-engineered nucleases coupled with a single guide RNA (gRNA) have been widely used to introduce site-specific DNA double-strand breaks (DSBs) 2,3 . The DSBs generated can be repaired either by (1) non-homologous/ microhomology-mediated end joining, often leading to insertion or deletion (indels) of some nucleotides at the break site and resulting in disruption of coding or regulatory function, or (2) homology-directed recombination (HDR), which exploits an exogenous DNA repair template with homologous sequences, resulting in gene replacement or insertion 4 .…”

mentioning

confidence: 99%

Genotoxic effects of base and prime editing in human hematopoietic stem cells

Fiumara,

Ferrari,

Omer-Javed

et al. 2023

Nat Biotechnol

Self Cite

View full text Add to dashboard Cite

Base and prime editors (BEs and PEs) may provide more precise genetic engineering than nuclease-based approaches because they bypass the dependence on DNA double-strand breaks. However, little is known about their cellular responses and genotoxicity. Here, we compared state-of-the-art BEs and PEs and Cas9 in human hematopoietic stem and progenitor cells with respect to editing efficiency, cytotoxicity, transcriptomic changes and on-target and genome-wide genotoxicity. BEs and PEs induced detrimental transcriptional responses that reduced editing efficiency and hematopoietic repopulation in xenotransplants and also generated DNA double-strand breaks and genotoxic byproducts, including deletions and translocations, at a lower frequency than Cas9. These effects were strongest for cytidine BEs due to suboptimal inhibition of base excision repair and were mitigated by tailoring delivery timing and editor expression through optimized mRNA design. However, BEs altered the mutational landscape of hematopoietic stem and progenitor cells across the genome by increasing the load and relative proportions of nucleotide variants. These findings raise concerns about the genotoxicity of BEs and PEs and warrant further investigation in view of their clinical application.

show abstract

mentioning

confidence: 99%

Genotoxic effects of base and prime editing in human hematopoietic stem cells

Fiumara,

Ferrari,

Omer-Javed

et al. 2023

Nat Biotechnol

Self Cite

View full text Add to dashboard Cite

show abstract

“…Growing evidence shows that a relevant fraction of cells treated with editing nucleases may experience large deletions at the targeted locus, translocations, chromosomal arm loss, and even chromothripsis (44,(55)(56)(57)(58). Nevertheless, assessing genome integrity using PCR-based sequencing methods might introduce amplification artifacts and should be complemented by multimodal innovative platforms, such as optical genome mapping, long-read sequencing, and CAST-seq (59)(60)(61).…”

Section: Discussionmentioning

confidence: 99%

“…However, increasing evidence has shown that the high burden of intact and fragmented AAV DNA triggers prolonged DDR affecting the repopulation potential and graft clonal diversity in xenograft models ( 33 , 44 , 64 – 66 ). Although AAVs has been considered a nonintegrating platform, integration of inverted terminal repeats (ITRs) or full-length AAV DNA in on- and off-target sites ( 44 , 67 – 69 ) raised concerns for the possible impact of ITR transcription promoting activity on genes flanking insertions ( 44 , 61 , 70 – 75 ). We moved to the IDLV platform and achieved in the most primitive HSPC subset similar HDR efficiency obtained with the AAV6 platform, indicating the feasibility and potential advantage of IDLV.…”

Section: Discussionmentioning

confidence: 99%

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

Castiello,

Brandas,

Ferrari

et al. 2024

Sci. Transl. Med.

Self Cite

View full text Add to dashboard Cite

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

show abstract

“…In principle, robust engraftment is an advantage of gene editing CD4+ T cells over HSPCs, whose repopulation capacity is instead usually hampered upon long-range gene editing. 11 , 20 In terms of QC, we developed assays to address product-specific potency, and theoretical immunogenicity concerns for residual Cas9, to meet recent indications by regulatory authorities.…”

Section: Discussionmentioning

confidence: 99%

Scalable GMP-compliant gene correction of CD4+ T cells with IDLV template functionally validated in vitro and in vivo

Asperti,

Canarutto,

Porcellini

et al. 2023

Molecular Therapy - Methods & Clinical Development

Self Cite

View full text Add to dashboard Cite

Genetic engineering meets hematopoietic stem cell biology for next-generation gene therapy

Cited by 30 publications

References 267 publications

Genotoxic effects of base and prime editing in human hematopoietic stem cells

Genotoxic effects of base and prime editing in human hematopoietic stem cells

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

Scalable GMP-compliant gene correction of CD4+ T cells with IDLV template functionally validated in vitro and in vivo

Contact Info

Product

Resources

About