Cas9 protein delivery non-integrating lentiviral vectors for gene correction in sickle cell disease

Uchida, Naoya; Drysdale, Claire; Nassehi, Tina; Gamer, Jackson; Yapundich, Morgan; DiNicola, Julia; Shibata, Yoshitaka; Hinds, Malikiya; Gudmundsdottir, Bjorg; Haro‐Mora, Juan J.; Demirci, Selami; Tisdale, John F.

doi:10.1016/j.omtm.2021.02.022

Cited by 35 publications

(17 citation statements)

References 56 publications

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“…The SCD mutation-targeting guide RNA allowed more specific editing for the bs-globin sequence, but less for the normal b-globin sequence, evaluated by a lentiviral delivery system. 16 For these early development studies, we used b-globin donor DNA that also included synonymous base pair changes that introduced a new HindIII enzyme site (6 bases). We detected $30% HDR-mediated HindIII site insertion in the bs-globin gene as evaluated by both enzyme digestion of polymerase chain reaction (PCR) products (Figure S1C) and targeted deep sequencing (Figure S1D).…”

Section: Therapeutic-level Gene Correction Of the Scd Mutation In Patient Cd34 + Cells With Electroporation Of Editing Toolsmentioning

confidence: 99%

Preclinical evaluation for engraftment of CD34+ cells gene-edited at the sickle cell disease locus in xenograft mouse and non-human primate models

Uchida

Nassehi

et al. 2021

Cell Reports Medicine

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Summary Sickle cell disease (SCD) is caused by a 20A > T mutation in the β-globin gene. Genome-editing technologies have the potential to correct the SCD mutation in hematopoietic stem cells (HSCs), producing adult hemoglobin while simultaneously eliminating sickle hemoglobin. Here, we developed high-efficiency viral vector-free non-footprint gene correction in SCD CD34 + cells with electroporation to deliver SCD mutation-targeting guide RNA, Cas9 endonuclease, and 100-mer single-strand donor DNA encoding intact β-globin sequence, achieving therapeutic-level gene correction at DNA (∼30%) and protein (∼80%) levels. Gene-edited SCD CD34 + cells contributed corrected cells 6 months post-xenograft mouse transplant without off-target δ-globin editing. We then developed a rhesus β-to-βs-globin gene conversion strategy to model HSC-targeted genome editing for SCD and demonstrate the engraftment of gene-edited CD34 + cells 10–12 months post-transplant in rhesus macaques. In summary, gene-corrected CD34 + HSCs are engraftable in xenograft mice and non-human primates. These findings are helpful in designing HSC-targeted gene correction trials.

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Section: Therapeutic-level Gene Correction Of the Scd Mutation In Patient Cd34 + Cells With Electroporation Of Editing Toolsmentioning

confidence: 99%

Preclinical evaluation for engraftment of CD34+ cells gene-edited at the sickle cell disease locus in xenograft mouse and non-human primate models

Uchida

Nassehi

et al. 2021

Cell Reports Medicine

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“…Recently, another study demonstrated that the NILV approach can overcome immune rejection and allow for the growth of transduced cells in an immunocompetent host by producing CRISPR-modified murine cell lines using mutated integrase vectors [ 137 ]. In addition to the advantages of the CRISPR/Cas9 system, another study proved that a Cas9 protein delivery system with NILVs encoding both guide RNA and donor DNA resulted in efficient DNA breakage, one-time genome correction of the sickle cell disease (SCD) mutation, and long-term engraftment of HSCs [ 126 ]. These studies explained the use of combined NILV approaches for a safer, long-lasting, and fruitful outcome in future research.…”

Section: Clinical Application Of Nilvsmentioning

confidence: 99%

Non-Integrating Lentiviral Vectors in Clinical Applications: A Glance Through

et al. 2022

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Lentiviral vectors (LVs) play an important role in gene therapy and have proven successful in clinical trials. LVs are capable of integrating specific genetic materials into the target cells and allow for long-term expression of the cDNA of interest. The use of non-integrating LVs (NILVs) reduces insertional mutagenesis and the risk of malignant cell transformation over integrating lentiviral vectors. NILVs enable transient expression or sustained episomal expression, especially in non-dividing cells. Important modifications have been made to the basic human immunodeficiency virus (HIV) structures to improve the safety and efficacy of LVs. NILV-aided transient expression has led to more pre-clinical studies on primary immunodeficiencies, cytotoxic cancer therapies, and hemoglobinopathies. Recently, the third generation of self-inactivating LVs was applied in clinical trials for recombinant protein production, vaccines, gene therapy, cell imaging, and induced pluripotent stem cell (iPSC) generation. This review discusses the basic lentiviral biology and the four systems used for generating NILV designs. Mutations or modifications in LVs and their safety are addressed with reference to pre-clinical studies. The detailed application of NILVs in promising pre-clinical studies is also discussed.

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“…IDLVs have been developed to prevent lentiviral integration into the targeted cell genome with efficient transduction [ 190 ]. IDLVs can deliver ZFN as well as CRISPR/Cas9 cargo to a broad range of dividing and quiescent tissues [ 191 , 192 ]. However, only a few studies of in vivo gene editing using LV delivery of CRISPR/Cas9 cargos have been reported in model animals.…”

Section: In Vivo Gene Editing For Scdmentioning

confidence: 99%

Hematopoietic Stem Cell Gene-Addition/Editing Therapy in Sickle Cell Disease

Germino-Watnick

Hinds

et al. 2022

Cells

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Autologous hematopoietic stem cell (HSC)-targeted gene therapy provides a one-time cure for various genetic diseases including sickle cell disease (SCD) and β-thalassemia. SCD is caused by a point mutation (20A > T) in the β-globin gene. Since SCD is the most common single-gene disorder, curing SCD is a primary goal in HSC gene therapy. β-thalassemia results from either the absence or the reduction of β-globin expression, and it can be cured using similar strategies. In HSC gene-addition therapy, patient CD34+ HSCs are genetically modified by adding a therapeutic β-globin gene with lentiviral transduction, followed by autologous transplantation. Alternatively, novel gene-editing therapies allow for the correction of the mutated β-globin gene, instead of addition. Furthermore, these diseases can be cured by γ-globin induction based on gene addition/editing in HSCs. In this review, we discuss HSC-targeted gene therapy in SCD with gene addition as well as gene editing.

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Cas9 protein delivery non-integrating lentiviral vectors for gene correction in sickle cell disease

Cited by 35 publications

References 56 publications

Preclinical evaluation for engraftment of CD34+ cells gene-edited at the sickle cell disease locus in xenograft mouse and non-human primate models

Preclinical evaluation for engraftment of CD34+ cells gene-edited at the sickle cell disease locus in xenograft mouse and non-human primate models

Non-Integrating Lentiviral Vectors in Clinical Applications: A Glance Through

Hematopoietic Stem Cell Gene-Addition/Editing Therapy in Sickle Cell Disease

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