Ex vivo cell-based CRISPR/Cas9 genome editing for therapeutic applications

As CRISPR-based therapies enter the clinic, evaluation of the safety remains a critical and still active area of study. While whole genome sequencing is an unbiased method for identifying somatic mutations introduced by ex vivo culture and genome editing, this methodology is unable to attain sufficient read depth to detect extremely low frequency events that could result in clonal expansion. As a solution, we utilized an exon capture panel to facilitate ultra-deep sequencing of >500 tumor suppressors and oncogenes most frequently altered in human cancer. We used this panel to investigate whether transient delivery of high-fidelity Cas9 protein targeted to three different loci (using guide RNAs (gRNAs) corresponding to sites at AAVS1, HBB, and ZFPM2) at day 4 and day 10 timepoints post-editing resulted in the introduction or enrichment of oncogenic mutations. In three separate primary human HSPC donors, we identified a mean of 1,488 variants per Cas9 treatment (at <0.07% limit of detection). After filtering to remove germline and/or synonymous changes, a mean of 3.3 variants remained per condition, which were further reduced to six total mutations after removing variants in unedited treatments. Of these, four variants resided at the predicted off-target site in the myelodysplasia-associated EZH2 gene that were subject to ZFPM2 gRNA targeting in Donors 2 and 3 at day 4 and day 10 timepoints. While Donor 1 displayed on-target cleavage at ZFPM2, we found no off-target activity at EZH2. Sanger sequencing revealed a homozygous single nucleotide polymorphism (SNP) at position 14bp distal from the Cas9 protospacer adjacent motif in EZH2 that eliminated any detectable off-target activity. We found no evidence of exonic off-target INDELs with either of the AAVS1 or HBB gRNAs. These findings indicate that clinically relevant delivery of high-fidelity Cas9 to primary HSPCs and ex vivo culture up to 10 days does not introduce or enrich for tumorigenic variants and that even a single SNP outside the seed region of the gRNA protospacer is sufficient to eliminate Cas9 off-target activity with this method of delivery into primary, repair competent human HSPCs.

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Optimization of AAV6 transduction enhances site-specific genome editing of primary human lymphocytes

10.1101/2021.05.03.440656 ◽

2021 ◽

Author(s):

Geoffrey L. Rogers ◽

Chun Huang ◽

Robert Clark ◽

Eduardo Seclen ◽

Hsu-Yu Chen ◽

...

Keyword(s):

B Cells ◽

Genome Editing ◽

Ex Vivo ◽

Human Lymphocytes ◽

Hematopoietic Cells ◽

Culture Conditions ◽

Adeno Associated Virus ◽

Optimized Protocol ◽

Virus Serotype ◽

Donor Template

Adeno-associated virus serotype 6 (AAV6) is a valuable reagent for genome editing of hematopoietic cells due to its ability to serve as a homology donor template. However, a comprehensive study of AAV6 transduction of hematopoietic cells in culture, with the goal of maximizing ex vivo genome editing, has not been reported. Here, we evaluated how the presence of serum, culture volume, transduction time, and electroporation parameters could influence AAV6 transduction. Based on these results, we identified an optimized protocol for genome editing of human lymphocytes based on a short, highly concentrated AAV6 transduction in the absence of serum, followed by electroporation with a targeted nuclease. In human CD4+ T cells and B cells, this protocol improved editing rates up to 7-fold and 21-fold respectively, when compared to standard AAV6 transduction protocols described in the literature. As a result, editing frequencies could be maintained using 50-100-fold less AAV6, which also reduced cellular toxicity. Our results highlight the important contribution of cell culture conditions for ex vivo genome editing with AAV6 vectors and provide a blueprint for improving AAV6-mediated homology-directed editing of human T and B cells.

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High-Efficiency Transduction of Primary Human Hematopoietic Stem/Progenitor Cells By AAV6 Vectors: strategies for Overcoming Donor-Variation and Implications in Genome Editing

Blood ◽

10.1182/blood.v128.22.5889.5889 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5889-5889

Author(s):

Chen Ling ◽

Kanit Bhukhai ◽

Zifei Yin ◽

Mengqun Tan ◽

Mervin Yoder ◽

...

Keyword(s):

Progenitor Cells ◽

Genome Editing ◽

High Efficiency ◽

Ex Vivo ◽

Transduction Efficiency ◽

Triple Mutant ◽

Hematopoietic Stem ◽

Combined Use ◽

Donor Variation ◽

Viral Capsid Proteins

Abstract We have reported that of the 10 commonly used AAV serotype vectors, AAV6 is the most efficient in transducing primary human hematopoietic stem/progenitor cells (HSPCs). However, the transduction efficiency of the wild-type (WT) AAV6 vector varies greatly in HSPCs from different donors. Here we report two distinct strategies to further increase the transduction efficiency in HSPCs from donors that are transduced poorly with the WT AAV6 vectors. The first strategy involved modification of the viral capsid proteins where specific surface-exposed tyrosine (Y) and threonine (T) residues were mutagenized to generate a triple-mutant (Y705+Y731F+T492V) AAV6 vector. The second strategy involved the use of ex vivo transduction at high cell density, which revealed a novel mechanism, which we have termed, 'cross-transduction'. The combined use of these strategies resulted in transduction efficiency exceeding ~90% in HSPCs. Our studies have significant implications in the optimal use of capsid-optimized AAV6 vectors in genome editing in HSPCs. Disclosures Leboulch: bluebird bio: Patents & Royalties. Payen:bluebird bio: Patents & Royalties. Srivastava:AGTC; Genzyme: Patents & Royalties.

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Reactivation of γ-globin in adult β-YAC mice after ex vivo and in vivo hematopoietic stem cell genome editing

Blood ◽

10.1182/blood-2018-03-838540 ◽

2018 ◽

Vol 131 (26) ◽

pp. 2915-2928 ◽

Cited By ~ 23

Author(s):

Chang Li ◽

Nikoletta Psatha ◽

Pavel Sova ◽

Sucheol Gil ◽

Hongjie Wang ◽

...

Keyword(s):

Gene Therapy ◽

Stem Cell ◽

Binding Site ◽

Genome Editing ◽

Ex Vivo ◽

Hematopoietic Stem ◽

Key Points ◽

Cell Genome ◽

Hsc Transplantation

Key Points CRISPR/Cas9-mediated disruption of a BCL11A binding site in HSCs of β-YAC mice results in the reactivation of γ-globin in erythrocytes. Our approach for in vivo HSC genome editing that does not require HSC transplantation and myeloablation should simplify HSC gene therapy.

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Genome-Editing and Biomedical Cell Products: Current State, Safety and Efficacy

BIOpreparations Prevention Diagnosis Treatment ◽

10.30895/2221-996x-2018-18-3-140-149 ◽

2018 ◽

Vol 18 (3) ◽

pp. 140-149 ◽

Cited By ~ 1

Author(s):

A. A. Goryaev ◽

M. V. Savkina ◽

K. M. Mefed ◽

V. P. Bondarev ◽

V. A. Merkulov ◽

...

Keyword(s):

T Cells ◽

Genome Editing ◽

Ex Vivo ◽

Rapid Development ◽

The United States ◽

Human Cells ◽

The European Union ◽

Antigen Receptors ◽

Current State ◽

Cd34 Cells

Advances in ex vivo technologies of human genome editing have made it possible to develop new approaches to the treatment of genetic, oncological, infectious and other diseases, which may involve the use of biomedical cell products. However, despite the rapid development of these technologies and a large number of clinical trials conducted in many countries around the world, only 4 products (Strimvelis, Zalmoxis, Kymriah and Yescarta) containing ex vivo genetically modified human cells are authorised for use in the European Union and the United States of America. This paper considers three promising technologies (ZFN, TALEN and CRISPR) that allow for easy and effective editing of the genome at the sites of interest, thereby creating a platform for further development of the genetic engineering of human cells. It describes the technology of engineering chimeric antigen receptors (CARs). It also provides data on the efficacy and safety of the approved products: Strimvelis which contains autologous CD34+ cells transduced ex vivo with a retroviral vector containing adenosine deaminase gene, Zalmoxis which contains modified allogeneic T-cells, and two products: Kymriah and Yescarta which contain autologous T-cells with CARs to CD19 antigen, intended for the treatment of CD19+ hematological malignancies.

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In vivo CRISPR-Cas gene editing with no detectable genome-wide off-target mutations

10.1101/272724 ◽

2018 ◽

Cited By ~ 4

Author(s):

Pinar Akcakaya ◽

Maggie L. Bobbin ◽

Jimmy A. Guo ◽

Jose M. Lopez ◽

M. Kendell Clement ◽

...

Keyword(s):

Genome Editing ◽

Ex Vivo ◽

Strong Support ◽

Guide Rna ◽

Genome Wide ◽

Highly Sensitive ◽

Further Development ◽

Target Effects ◽

Cas Gene

CRISPR-Cas genome-editing nucleases hold substantial promise for human therapeutics1–5 but identifying unwanted off-target mutations remains an important requirement for clinical translation6, 7. For ex vivo therapeutic applications, previously published cell-based genome-wide methods provide potentially useful strategies to identify and quantify these off-target mutation sites8–12. However, a well-validated method that can reliably identify off-targets in vivo has not been described to date, leaving the question of whether and how frequently these types of mutations occur. Here we describe Verification of In Vivo Off-targets (VIVO), a highly sensitive, unbiased, and generalizable strategy that we show can robustly identify genome-wide CRISPR-Cas nuclease off-target effects in vivo. To our knowledge, these studies provide the first demonstration that CRISPR-Cas nucleases can induce substantial off-target mutations in vivo, a result we obtained using a deliberately promiscuous guide RNA (gRNA). More importantly, we used VIVO to show that appropriately designed gRNAs can direct efficient in vivo editing without inducing detectable off-target mutations. Our findings provide strong support for and should encourage further development of in vivo genome editing therapeutic strategies.

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