Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia.

The absence of cancer-restricted surface markers is a major impediment to antigen-specific immunotherapy using chimeric antigen receptor (CAR) T cells. For example, targeting the canonical myeloid marker CD33 in acute myeloid leukemia (AML) results in toxicity from destruction of normal myeloid cells. We hypothesized that a leukemia-specific antigen could be created by deleting CD33 from normal hematopoietic stem and progenitor cells (HSPCs), thereby generating a hematopoietic system resistant to CD33-targeted therapy and enabling specific targeting of AML with CAR T cells. We generated CD33-deficient human HSPCs and demonstrated normal engraftment and differentiation in immunodeficient mice. Autologous CD33 KO HSPC transplantation in rhesus macaques demonstrated long-term multilineage engraftment of gene-edited cells with normal myeloid function. CD33-deficient cells were impervious to CD33-targeting CAR T cells, allowing for efficient elimination of leukemia without myelotoxicity. These studies illuminate a novel approach to antigen-specific immunotherapy by genetically engineering the host to avoid on-target, off-tumor toxicity.

Selective haematological cancer eradication with preserved haematopoiesis.

Haematopoietic stem cell (HSC) transplantation (HSCT) is the only curative treatment for a broad range of haematological malignancies, but the standard of care relies on untargeted chemotherapies and limited possibilities to treat malignant cells after HSCT without affecting the transplanted healthy cells1. Antigen-specific cell-depleting therapies hold the promise of much more targeted elimination of diseased cells, as witnessed in the past decade by the revolution of clinical practice for B cell malignancies2. However, target selection is complex and limited to antigens expressed on subsets of haematopoietic cells, resulting in a fragmented therapy landscape with high development costs2-5. Here we demonstrate that an antibody-drug conjugate (ADC) targeting the pan-haematopoietic marker CD45 enables the antigen-specific depletion of the entire haematopoietic system, including HSCs. Pairing this ADC with the transplantation of human HSCs engineered to be shielded from the CD45-targeting ADC enables the selective eradication of leukaemic cells with preserved haematopoiesis. The combination of CD45-targeting ADCs and engineered HSCs creates an almost universal strategy to replace a diseased haematopoietic system, irrespective of disease aetiology or originating cell type. We propose that this approach could have broad implications beyond haematological malignancies.

Base editing of haematopoietic stem cells rescues sickle cell disease in mice.

Sickle cell disease (SCD) is caused by a mutation in the ß-globin gene HBB1. We used a custom adenine base editor (ABE8e-NRCH)2,3 to convert the SCD allele (HBBS) into Makassar ß-globin (HBBG), a non-pathogenic variant4,5. Ex vivo delivery of mRNA encoding the base editor with a targeting guide RNA into haematopoietic stem and progenitor cells (HSPCs) from patients with SCD resulted in 80% conversion of HBBS to HBBG. Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice, the frequency of HBBG was 68% and hypoxia-induced sickling of bone marrow reticulocytes had decreased fivefold, indicating durable gene editing. To assess the physiological effects of HBBS base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse6 and then transplanted these cells into irradiated mice. After sixteen weeks, Makassar ß-globin represented 79% of ß-globin protein in blood, and hypoxia-induced sickling was reduced threefold. Mice that received base-edited HSPCs showed near-normal haematological parameters and reduced splenic pathology compared to mice that received unedited cells. Secondary transplantation of edited bone marrow confirmed that the gene editing was durable in long-term haematopoietic stem cells and showed that HBBS-to-HBBG editing of 20% or more is sufficient for phenotypic rescue. Base editing of human HSPCs avoided the p53 activation and larger deletions that have been observed following Cas9 nuclease treatment. These findings point towards a one-time autologous treatment for SCD that eliminates pathogenic HBBS, generates benign HBBG, and minimizes the undesired consequences of double-strand DNA breaks.

The NIH Somatic Cell Genome Editing program.

The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium's plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled-along with validated datasets-into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit-and the knowledge generated by its applications-as a means to accelerate the clinical development of new therapies for a wide range of conditions.

Development and IND-enabling studies of a novel Cas9 genome-edited autologous CD34+ cell therapy to induce fetal hemoglobin for sickle cell disease.

Sickle cell disease (SCD) is a common, severe genetic blood disorder. Current pharmacotherapies are partially effective and allogeneic hematopoietic stem cell transplantation is associated with immune toxicities. Genome editing of patient hematopoietic stem cells (HSCs) to reactivate fetal hemoglobin (HbF) in erythroid progeny offers an alternative potentially curative approach to treat SCD. Although the FDA released guidelines for evaluating genome editing risks, it remains unclear how best to approach pre-clinical assessment of genome-edited cell products. Here, we describe rigorous pre-clinical development of a therapeutic γ-globin gene promoter editing strategy that supported an investigational new drug application cleared by the FDA. We compared γ-globin promoter and BCL11A enhancer targets, identified a potent HbF-inducing lead candidate, and tested our approach in mobilized CD34+ hematopoietic stem progenitor cells (HSPCs) from SCD patients. We observed efficient editing, HbF induction to predicted therapeutic levels, and reduced sickling. With single-cell analyses, we defined the heterogeneity of HbF induction and HBG1/HBG2 transcription. With CHANGE-seq for sensitive and unbiased off-target discovery followed by targeted sequencing, we did not detect off-target activity in edited HSPCs. Our study provides a blueprint for translating new ex vivo HSC genome editing strategies toward clinical trials for treating SCD and other blood disorders.

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

CRISPR-Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications1-6 but identifying unwanted off-target mutations is important for clinical translation7. A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe 'verification of in vivo off-targets' (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR-Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR-Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing.

CRISPR-targeted MAGT1 insertion restores XMEN patient hematopoietic stem cells and lymphocytes.

XMEN disease, defined as "X-linked MAGT1 deficiency with increased susceptibility to Epstein-Barr virus infection and N-linked glycosylation defect," is a recently described primary immunodeficiency marked by defective T cells and natural killer (NK) cells. Unfortunately, a potentially curative hematopoietic stem cell transplantation is associated with high mortality rates. We sought to develop an ex vivo targeted gene therapy approach for patients with XMEN using a CRISPR/Cas9 adeno-associated vector (AAV) to insert a therapeutic MAGT1 gene at the constitutive locus under the regulation of the endogenous promoter. Clinical translation of CRISPR/Cas9 AAV-targeted gene editing (GE) is hampered by low engraftable gene-edited hematopoietic stem and progenitor cells (HSPCs). Here, we optimized GE conditions by transient enhancement of homology-directed repair while suppressing AAV-associated DNA damage response to achieve highly efficient (>60%) genetic correction in engrafting XMEN HSPCs in transplanted mice. Restored MAGT1 glycosylation function in human NK and CD8+ T cells restored NK group 2 member D (NKG2D) expression and function in XMEN lymphocytes for potential treatment of infections, and it corrected HSPCs for long-term gene therapy, thus offering 2 efficient therapeutic options for XMEN poised for clinical translation.

Enhanced homology-directed repair for highly efficient gene editing in hematopoietic stem/progenitor cells.

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 Streptococcus pyogenes Cas9 mRNA and an oligodeoxynucleotide donor to repair genetic mutations showed the capability to restore physiological protein expression but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we report that transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology-directed repair to achieve highly efficient (80% gp91phox+ cells compared with healthy donor control subjects) long-term correction of X-CGD CD34+ cells.

Prediction and validation of hematopoietic stem and progenitor cell off-target editing in transplanted rhesus macaques.

The programmable nuclease technology CRISPR-Cas9 has revolutionized gene editing in the last decade. Due to the risk of off-target editing, accurate and sensitive methods for off-target characterization are crucial prior to applying CRISPR-Cas9 therapeutically. Here, we utilized a rhesus macaque model to compare the predictive values of CIRCLE-seq, an in vitro off-target prediction method, with in silico prediction (ISP) based solely on genomic sequence comparisons. We use AmpliSeq HD error-corrected sequencing to validate off-target sites predicted by CIRCLE-seq and ISP for a CD33 guide RNA (gRNA) with thousands of off-target sites predicted by ISP and CIRCLE-seq. We found poor correlation between the sites predicted by the two methods. When almost 500 sites predicted by each method were analyzed by error-corrected sequencing of hematopoietic cells following transplantation, 19 off-target sites revealed insertion or deletion mutations. Of these sites, 8 were predicted by both methods, 8 by CIRCLE-seq only, and 3 by ISP only. The levels of cells with these off-target edits exhibited no expansion or abnormal behavior in vivo in animals followed for up to 2 years. In addition, we utilized an unbiased method termed CAST-seq to search for translocations between the on-target site and off-target sites present in animals following transplantation, detecting one specific translocation that persisted in blood cells for at least 1 year following transplantation. In conclusion, neither CIRCLE-seq or ISP predicted all sites, and a combination of careful gRNA design, followed by screening for predicted off-target sites in target cells by multiple methods, may be required for optimizing safety of clinical development.

Defining and improving the genome-wide specificities of CRISPR-Cas9 nucleases.

CRISPR-Cas9 RNA-guided nucleases are a transformative technology for biology, genetics and medicine owing to the simplicity with which they can be programmed to cleave specific DNA target sites in living cells and organisms. However, to translate these powerful molecular tools into safe, effective clinical applications, it is of crucial importance to carefully define and improve their genome-wide specificities. Here, we outline our state-of-the-art understanding of target DNA recognition and cleavage by CRISPR-Cas9 nucleases, methods to determine and improve their specificities, and key considerations for how to evaluate and reduce off-target effects for research and therapeutic applications.

High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.

CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.

Systematic screening reveals a role for BRCA1 in the response to transcription-associated DNA damage.

BRCA1 is a breast and ovarian tumor suppressor. Given its numerous incompletely understood functions and the possibility that more exist, we performed complementary systematic screens in search of new BRCA1 protein-interacting partners. New BRCA1 functions and/or a better understanding of existing ones were sought. Among the new interacting proteins identified, genetic interactions were detected between BRCA1 and four of the interactors: TONSL, SETX, TCEANC, and TCEA2. Genetic interactions were also detected between BRCA1 and certain interactors of TONSL, including both members of the FACT complex. From these results, a new BRCA1 function in the response to transcription-associated DNA damage was detected. Specifically, new roles for BRCA1 in the restart of transcription after UV damage and in preventing or repairing damage caused by stabilized R loops were identified. These roles are likely carried out together with some of the newly identified interactors. This new function may be important in BRCA1 tumor suppression, since the expression of several interactors, including some of the above-noted transcription proteins, is repeatedly aberrant in both breast and ovarian cancers.

Zebrafish dscaml1 Deficiency Impairs Retinal Patterning and Oculomotor Function.

Down syndrome cell adhesion molecules (dscam and dscaml1) are essential regulators of neural circuit assembly, but their roles in vertebrate neural circuit function are still mostly unexplored. We investigated the functional consequences of dscaml1 deficiency in the larval zebrafish (sexually undifferentiated) oculomotor system, where behavior, circuit function, and neuronal activity can be precisely quantified. Genetic perturbation of dscaml1 resulted in deficits in retinal patterning and light adaptation, consistent with its known roles in mammals. Oculomotor analyses revealed specific deficits related to the dscaml1 mutation, including severe fatigue during gaze stabilization, reduced saccade amplitude and velocity in the light, greater disconjugacy, and impaired fixation. Two-photon calcium imaging of abducens neurons in control and dscaml1 mutant animals confirmed deficits in saccade-command signals (indicative of an impairment in the saccadic premotor pathway), whereas abducens activation by the pretectum-vestibular pathway was not affected. Together, we show that loss of dscaml1 resulted in impairments in specific oculomotor circuits, providing a new animal model to investigate the development of oculomotor premotor pathways and their associated human ocular disorders.SIGNIFICANCE STATEMENTDscaml1 is a neural developmental gene with unknown behavioral significance. Using the zebrafish model, this study shows that dscaml1 mutants have a host of oculomotor (eye movement) deficits. Notably, the oculomotor phenotypes in dscaml1 mutants are reminiscent of human ocular motor apraxia, a neurodevelopmental disorder characterized by reduced saccade amplitude and gaze stabilization deficits. Population-level recording of neuronal activity further revealed potential subcircuit-specific requirements for dscaml1 during oculomotor behavior. These findings underscore the importance of dscaml1 in the development of visuomotor function and characterize a new model to investigate potential circuit deficits underlying human oculomotor disorders.

Engineered CRISPR-Cas9 nucleases with altered PAM specificities.

Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.

CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets.

Sensitive detection of off-target effects is important for translating CRISPR-Cas9 nucleases into human therapeutics. In vitro biochemical methods for finding off-targets offer the potential advantages of greater reproducibility and scalability while avoiding limitations associated with strategies that require the culture and manipulation of living cells. Here we describe circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), a highly sensitive, sequencing-efficient in vitro screening strategy that outperforms existing cell-based or biochemical approaches for identifying CRISPR-Cas9 genome-wide off-target mutations. In contrast to previously described in vitro methods, we show that CIRCLE-seq can be practiced using widely accessible next-generation sequencing technology and does not require reference genome sequences. Importantly, CIRCLE-seq can be used to identify off-target mutations associated with cell-type-specific single-nucleotide polymorphisms, demonstrating the feasibility and importance of generating personalized specificity profiles. CIRCLE-seq provides an accessible, rapid, and comprehensive method for identifying genome-wide off-target mutations of CRISPR-Cas9.

Continuous directed evolution of DNA-binding proteins to improve TALEN specificity.

Nucleases containing programmable DNA-binding domains can alter the genomes of model organisms and have the potential to become human therapeutics. Here we present DNA-binding phage-assisted continuous evolution (DB-PACE) as a general approach for the laboratory evolution of DNA-binding activity and specificity. We used this system to generate transcription activator-like effectors nucleases (TALENs) with broadly improved DNA cleavage specificity, establishing DB-PACE as a versatile approach for improving the accuracy of genome-editing agents.

Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity.

Although transcription activator-like effector nucleases (TALENs) can be designed to cleave chosen DNA sequences, TALENs have activity against related off-target sequences. To better understand TALEN specificity, we profiled 30 unique TALENs with different target sites, array length and domain sequences for their abilities to cleave any of 10(12) potential off-target DNA sequences using in vitro selection and high-throughput sequencing. Computational analysis of the selection results predicted 76 off-target substrates in the human genome, 16 of which were accessible and modified by TALENs in human cells. The results suggest that (i) TALE repeats bind DNA relatively independently; (ii) longer TALENs are more tolerant of mismatches yet are more specific in a genomic context; and (iii) excessive DNA-binding energy can lead to reduced TALEN specificity in cells. Based on these findings, we engineered a TALEN variant that exhibits equal on-target cleavage activity but tenfold lower average off-target activity in human cells.

Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs.

Transcription activator-like effector nucleases (TALENs) are powerful new research tools that enable targeted gene disruption in a wide variety of model organisms. Recent work has shown that TALENs can induce mutations in endogenous zebrafish genes, but to date only four genes have been altered, and larger-scale tests of the success rate, mutation efficiencies and germline transmission rates have not been described. Here, we constructed homodimeric TALENs to 10 different targets in various endogenous zebrafish genes and found that 7 nuclease pairs induced targeted indel mutations with high efficiencies ranging from 2 to 76%. We also tested obligate heterodimeric TALENs and found that these nucleases induce mutations with comparable or higher frequencies and have better toxicity profiles than their homodimeric counterparts. Importantly, mutations induced by both homodimeric and heterodimeric TALENs are passed efficiently through the germline, in some cases reaching 100% transmission. For one target gene sequence, we observed substantially reduced mutagenesis efficiency for a variant site bearing two mismatched nucleotides, raising the possibility that TALENs might be used to perform allele-specific gene disruption. Our results suggest that construction of one to two heterodimeric TALEN pairs for any given gene will, in most cases, enable researchers to rapidly generate knockout zebrafish.

CHANGE-seq-BE enables simultaneously sensitive and unbiased in vitro profiling of base editor genome-wide activity.

Base editors ( BE ) enable programmable conversion of nucleotides in genomic DNA without double-stranded breaks and have substantial promise to become new transformative genome editing medicines. Sensitive and unbiased detection of base editor off-target effects is important for identifying safety risks unique to base editors and translation to human therapeutics, as well as accurate use in life sciences research. However, current methods for understanding the global activities of base editors have limitations in terms of sensitivity or bias. Here we present CHANGE-seq-BE, a novel method to directly assess the off-target profile of base editors that is simultaneously sensitive and unbiased. CHANGE-seq-BE is based on the principle of selective sequencing of adenine base editor modified genomic DNA in vitro , and provides an accessible, rapid, and comprehensive method for identifying genome-wide off-target mutations of base editors.