Efficient Delivery and Nuclear Uptake Is Not Sufficient to Detect Gene Editing in CD34+ Cells Directed by a Ribonucleoprotein Complex

While there are several small molecule, gene therapy, and gene editing approaches for treating sickle cell disease (SCD), these strategies do not result in the direct elimination of the causative sickle β-globin (HbS) variant itself. The reduction or complete removal of this pathologic globin variant and expression of normal β-hemoglobin (HbB) or other non-polymerizing β-globin variant may increase the likelihood of beneficial outcomes for SCD patients. Adenine base editors (ABEs) can precisely convert the mutant A-T base pair responsible for SCD to a G-C base pair, thus generating the hemoglobin variant, Hb G-Makassar, a naturally occurring β-globin variant that is not associated with human disease. Our studies have identified ABEs that can achieve highly efficient Makassar editing (>70%) of the sickle mutation in both sickle trait (HbAS) and homozygous sickle (HbSS) patient CD34+ cells with high cell viability and recovery and without perturbation of immunophenotypic hematopoietic stem and progenitor cell (HSPC) frequencies after ex vivo delivery of guide RNA and mRNA encoding the ABE. Furthermore, Makassar editing was retained throughout erythropoiesis in bulk in vitro erythroid differentiated cells (IVED) derived from edited CD34+ cells. To gain an understanding of allelic editing at a single clone resolution, we assessed editing frequencies of clones from both single cell expansion in erythroid differentiation media, as well as from single BFU-E colonies. We found that we could achieve >70% of colonies with bi-allelic Makassar editing and approximately 20% of colonies with mono-allelic Makassar editing, while <3% of colonies remained completely unedited. Previously, conventional hemoglobin capillary electrophoresis and high-performance liquid chromatography (HPLC) were unable to distinguish between HbS and HbG-Makassar. Here, we developed an ultra-high-performance liquid chromatography (UPLC) method that resolves sickle globin (HbS) from Hb G-Makassar globin in IVED cells. The Makassar globin variant was further confirmed by liquid chromatography mass spectrometry (LC-MS). By developing this new method to resolve these two β-globin variants in edited HbSS cells, we were able to detect, in bulk IVED cultures, >80% abundance Hb G-Makassar of total β-globins, which corresponded to a concomitant reduction of HbS levels to <20%. Furthermore, we were also able to determine globin abundance as well as allelic editing at the level of single clones and found that HbS was completely eliminated in >70% of cells that had bi-allelic Makassar editing. Moreover, in the approximately 20% of colonies that were found to be mono-allelically edited for the Makassar variant, there was a 60:40 ratio of Hb G-Makassar:HbS globin abundance in individual clones, at levels remarkably similar to the HbA(wildtype HbB):HbS levels found in HbAS individuals, with minimal observable in vitro sickling when exposed to hypoxia. Thus, with our ABEs, we were able to reduce HbS to <40% on a per cell basis in >90% of IVED cells and found that in vitro sickling under hypoxia inversely correlated with the level of Hb G-Makassar globin variant installation and corresponding reduction in HbS levels. By converting HbS to Hb G-Makassar, our direct and precise editing strategy replaces a pathogenic β-globin with one that has been shown to have normal hematologic parameters. Coupled with autologous stem cell transplant, this next generation gene editing strategy presents a promising new modality for treating patients with SCD. Disclosures Chu: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lam:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Packer:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Olins:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Liquori:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Marshall:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lee:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Yan:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Decker:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gantzer:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Haskett:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Bohnuud:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Born:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Barrera:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Slaymaker:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gaudelli:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hartigan:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ciaramella:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company.

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Peptide Brush Polymers for Efficient Delivery of a Gene Editing Protein to Stem Cells

Angewandte Chemie International Edition ◽

10.1002/anie.201904894 ◽

2019 ◽

Vol 58 (44) ◽

pp. 15646-15649 ◽

Cited By ~ 9

Author(s):

Angela P. Blum ◽

David A. Nelles ◽

Francisco J. Hidalgo ◽

Mollie A. Touve ◽

Deborah S. Sim ◽

...

Keyword(s):

Stem Cells ◽

Gene Editing ◽

Efficient Delivery ◽

Brush Polymers

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Precise and error-prone CRISPR-directed gene editing activity in human CD34+ cells varies widely among patient samples

Gene Therapy ◽

10.1038/s41434-020-00192-z ◽

2020 ◽

Author(s):

Shirin R. Modarai ◽

Sambee Kanda ◽

Kevin Bloh ◽

Lynn M. Opdenaker ◽

Eric B. Kmiec

Keyword(s):

Gene Editing ◽

Human Cd34 ◽

Cd34 Cells ◽

Editing Activity

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CRISPR/Cas9-loaded stealth liposomes effectively cleared established HPV16-driven tumours in syngeneic mice

PLoS ONE ◽

10.1371/journal.pone.0223288 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0223288

Author(s):

Luqman Jubair ◽

Alfred K. Lam ◽

Sora Fallaha ◽

Nigel A. J. McMillan

Keyword(s):

Cell Death ◽

Gene Editing ◽

Cancer Cell Line ◽

Host Immune System ◽

Stealth Liposomes ◽

Efficient Delivery ◽

Significant Toxicity ◽

Immunocompetent Mice ◽

First Time

Gene-editing has raised the possibility of being able to treat or cure cancers, but key challenges remain, including efficient delivery, in vivo efficacy, and its safety profile. Ideal targets for cancer therapy are oncogenes, that when edited, cause cell death. Here, we show, using the human papillomavirus (HPV) type 16 cancer cell line TC1, that CRISPR/Cas9 targeting the E7 oncogene and packaged in PEGylated liposomes cleared established tumours in immunocompetent mice. Treatment caused no significant toxicity in the spleen or liver. An ideal therapeutic outcome would be the induction of an immunogenic cell death (ICD), such that recurrent tumours would be eliminated by the host immune system. We show here for the first time that CRISPR/Cas9-mediated cell death via targeting E7 did not result in ICD. Overall, our data show that in vivo CRISPR/Cas targeting of oncogenes is an effective treatment approach for cancer.

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Adapting CRISPR/Cas9 System for Targeting Mitochondrial Genome

Frontiers in Genetics ◽

10.3389/fgene.2021.627050 ◽

2021 ◽

Vol 12 ◽

Author(s):

Syed-Rehan A. Hussain ◽

Mehmet E. Yalvac ◽

Benedict Khoo ◽

Sigrid Eckardt ◽

K. John McLaughlin

Keyword(s):

Mitochondrial Genome ◽

Gene Editing ◽

Stem Loop ◽

Guide Rna ◽

Novel Approach ◽

Nadh Ubiquinone Oxidoreductase ◽

Efficient Delivery ◽

Localization Sequence ◽

Loop Element ◽

Enzyme Complexes

Gene editing of the mitochondrial genome using the CRISPR-Cas9 system is highly challenging mainly due to sub-efficient delivery of guide RNA and Cas9 enzyme complexes into the mitochondria. In this study, we were able to perform gene editing in the mitochondrial DNA by appending an NADH-ubiquinone oxidoreductase chain 4 (ND4) targeting guide RNA to an RNA transport-derived stem loop element (RP-loop) and expressing the Cas9 enzyme with a preceding mitochondrial localization sequence. We observe mitochondrial colocalization of RP-loop gRNA and a marked reduction of ND4 expression in the cells carrying a 11205G variant in their ND4 sequence coincidently decreasing the mtDNA levels. This proof-of-concept study suggests that a stem-loop element added sgRNA can be transported to the mitochondria and functionally interact with Cas9 to mediate sequence-specific mtDNA cleavage. Using this novel approach to target the mtDNA, our results provide further evidence that CRISPR-Cas9-mediated gene editing might potentially be used to treat mitochondrial-related diseases.

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Advances in delivery vectors for gene therapy in liver cancer

Therapeutic Delivery ◽

10.4155/tde-2019-0076 ◽

2020 ◽

Vol 11 (1) ◽

pp. 833-850 ◽

Cited By ~ 5

Author(s):

Katherine E Redd Bowman ◽

Phong Lu ◽

Erica R Vander Mause ◽

Carol S Lim

Keyword(s):

Hepatocellular Carcinoma ◽

Gene Therapy ◽

Viral Vectors ◽

Gene Editing ◽

Cancer Death ◽

Common Cause ◽

The Third ◽

Gene Delivery Vectors ◽

Efficient Delivery ◽

Therapy Delivery

Hepatocellular carcinoma (HCC) is the third most common cause of cancer death globally, mainly due to lack of effective treatments – a problem that gene therapy is poised to solve. Successful gene therapy requires safe and efficient delivery vectors, and recent advances in both viral and nonviral vectors have made an important impact on HCC gene therapy delivery. This review explores how adenoviral, retroviral and adeno-associated viral vectors have been modified to increase safety and delivery capacity, highlighting studies and clinical trials using these vectors for HCC gene therapy. Nanoparticles, liposomes, exosomes and virosomes are also featured in their roles as HCC gene delivery vectors. Finally, new discoveries in gene editing technology and their impacts on HCC gene therapy are discussed.

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Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM

Cells ◽

10.3390/cells10082099 ◽

2021 ◽

Vol 10 (8) ◽

pp. 2099

Author(s):

Yunxing Liu ◽

Fang Liang ◽

Zijiong Dong ◽

Song Li ◽

Jianmin Ye ◽

...

Keyword(s):

Genome Editing ◽

Streptococcus Pyogenes ◽

Gene Editing ◽

Zebrafish Genome ◽

Human Cells ◽

Ribonucleoprotein Complex ◽

Guide Rna ◽

Protospacer Adjacent Motif ◽

Low Activity

The CRISPR/Cas9 system has been widely used for gene editing in zebrafish. However, the required NGG protospacer adjacent motif (PAM) of Streptococcus pyogenes Cas9 (SpCas9) notably restricts the editable range of the zebrafish genome. Recently, Cas9 from S. canis (ScCas9), which has a more relaxed 5′-NNG-3′ PAM, was reported to have activities in human cells and plants. However, the editing ability of ScCas9 has not been tested in zebrafish. Here we characterized and optimized the activity of ScCas9 in zebrafish. Delivered as a ribonucleoprotein complex, ScCas9 can induce mutations in zebrafish. Using the synthetic modified crRNA:tracrRNA duplex instead of in vitro-transcribed single guide RNA, the low activity at some loci were dramatically improved in zebrafish. As far as we know, our work is the first report on the evaluation of ScCas9 in animals. Our work optimized ScCas9 as a new nuclease for targeting relaxed NNG PAMs for zebrafish genome editing, which will further improve genome editing in zebrafish.

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Gene Editing and mRNA-Based Therapy: Two Complementary Therapeutic Approaches for the Treatment of Patients with Xmen Disease

Blood ◽

10.1182/blood-2019-129062 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4637-4637

Author(s):

Julie Brault ◽

Ezekiel Bello ◽

Ronald J. Meis ◽

Taylor Liu ◽

Sherry M. Koontz ◽

...

Keyword(s):

T Cells ◽

Cytotoxic Activity ◽

Nk Cells ◽

Cd8 T Cells ◽

Adoptive Transfer ◽

Gene Editing ◽

Nkg2d Expression ◽

Cd34 Cells ◽

Cas9 Mrna

Introduction 'X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV) infection, and neoplasia' (XMEN) disease is a primary immunodeficiency disease caused by loss-of-function mutations in the MAGT1 gene encoding for the magnesium transporter 1. This leads to the absence of expression of the "Natural-Killer Group 2, member D" (NKG2D) receptor in natural killer (NK) and CD8+ T cells, which is essential for their antiviral and antitumoral cytotoxic activity. In consequence, XMEN patients develop chronic EBV infections and EBV-related lymphoproliferative disorders. Allogeneic bone marrow transplant has been associated with significant mortality, and there are no other effective treatments. In that context, we aimed at developing two complementary approaches to treat XMEN patients: 1) Adoptive transfer of XMEN T/NK cells corrected by transient mRNA therapy or longer-lasting gene editing therapy in order to control infections, and 2) Transplantation of gene-edited CD34+ cells in order to permanently restore production of functional immune cells. Material and methods CD34+ cells and PBMCs were collected from XMEN patients and healthy donors (HD) (NIH Protocol 94-I-073). For mRNA therapy, we expanded T cells with anti-CD3/anti-CD28 beads in RPMI + 10% serum supplemented with 200 IU/mL IL2 for 5-7 days and NK cells with 100 IU/mL IL2 and 10 ng/mL IL15 in culture with K562-mb15-41BBL for 10-15 days. Both XMEN T and NK cells were electroporated (EP) with MAGT1 mRNA and cultured for up to 28 days. For gene editing, XMEN CD34+ or stimulated T cells were electroporated with Cas9 mRNA and sgRNA; a rAAV6 donor encoding for the codon-optimized MAGT1 cDNA was added after EP. Two days post-EP, CD34+ cells were differentiated into NK cells for 35 days in vitro. Results MAGT1 mRNA-based therapy. We first showed a restored MAGT1 expression by western blot at 6h and 24h post-EP of the MAGT1 mRNA. In consequence, NKG2D expression analyzed by flow cytometry was restored in expanded CD8+ T and NK cells starting within the 6h post-EP (20-40%), with a peak at 48h (>85%) and a progressive decrease of the expression over time (still 40% and 75%, respectively, of CD8+ T and NK cells of cells at day 14 post-EP respectively). The cytotoxic activity of mRNA-corrected XMEN NK cells was analyzed by culture with K562 target cells at several effector:target (E:T) ratios and shown to be restored at a level similar to HD NK cells (mRNA-treated: 66.7% ±5.8%; HD: 67.8% ±5.9% at E:T 2:1 ratio) compared to untreated cells (49.0% ±7.2%) (Fig 1a). Anticipating the potential use of these cells for repeated infusions as a treatment modality to control infections, we demonstrated that MAGT1 mRNA-corrected CD8+ T and NK cells that have been cryopreserved and thawed exhibit the same NKG2D expression kinetics following thaw and culture. Gene editing therapy. XMEN CD34+ cells electroporated with Cas9 mRNA and a sgRNA targeting exon 1 of MAGT1 gene showed an in vitro average integration rate of the MAGT1 cDNA AAV donor of 35.6% (range: 33.8-41.9%). The NKG2D expression in AAV-treated CD34+-derived NK cells was approximatively of 23% (range: 14.2-27.9%). Interestingly, their cytotoxic activity was similar to the level of NKG2D expression (23.1% ±4.3%), significantly higher than in untreated cells (9.7% ±2.8%) (Fig 1b). Similar rates of targeted integration and NKG2D expression were also obtained in AAV-treated CD8+ T cells. Conclusion For the first time, we demonstrate the efficiency of two approaches for development of potential cell therapy treatments of XMEN patients. MAGT1 mRNA electroporation can restore efficient transient expression of NKG2D in CD8+ T and NK cells, thus fully restoring the cytotoxic activity of NK cells. In addition, cells electroporated with MAGT1 mRNA can be cryopreserved, thus allowing repeated infusions. In parallel, we showed that efficient targeted insertion can be achieved in CD8+ T cells and CD34+ cells by using an AAV donor although the level of NKG2D expression is lower. Optimizations are currently ongoing in order to reach higher levels of correction. Both approaches could be combined in order to propose a new therapeutic strategy for the treatment of XMEN patients: repetitive adoptive transfer of mRNA-corrected autologous T/NK cells for the prevention or control of intractable infections, and transplantation of gene-edited CD34+ cells for the definitive treatment of these patients. Disclosures Meis: CELLSCRIPT, LLC: Employment. Li:MaxCyte, Inc: Employment. Allen:MaxCyte, Inc: Employment. Clark:CELLSCRIPT, LLC: Employment. Dahl:CELLSCRIPT, LLC: Other: Owner and officer.

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Enhanced Homology-directed Repair for Highly Efficient Gene Editing in Hematopoietic Stem/Progenitor Cells

Blood ◽

10.1182/blood.2020008503 ◽

2021 ◽

Author(s):

Suk See De Ravin ◽

Julie Brault ◽

Ronald J Meis ◽

Siyuan Liu ◽

Linhong Li ◽

...

Keyword(s):

Gene Editing ◽

Hematopoietic Stem ◽

Vector Integration ◽

Homology Directed Repair ◽

Highly Efficient ◽

Donor Control ◽

Efficient Gene ◽

Cd34 Cells ◽

Viable Approach

Lentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a viable approach, but random vector integration and subnormal protein production from exogenous promoters in transduced cells remain concerning for long-term safety and efficacy. A previous genome editing-based approach using SpCas9 and an oligodeoxynucleotide donor to repair genetic mutations demonstrated the capability to restore physiological protein expression, but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we show transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology directed repair (HDR) to achieve highly efficient (80% gp91phox+ cells compared to healthy donor control) long-term correction of X-CGD CD34+ cells.

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