Enhancement of erythropoietic output by Cas9-mediated insertion of a natural variant in haematopoietic stem and progenitor cells.

Some gene polymorphisms can lead to monogenic diseases, whereas other polymorphisms may confer beneficial traits. A well-characterized example is congenital erythrocytosis-the non-pathogenic hyper-production of red blood cells-that is caused by a truncated erythropoietin receptor. Here we show that Cas9-mediated genome editing in CD34+ human haematopoietic stem and progenitor cells (HSPCs) can recreate the truncated form of the erythropoietin receptor, leading to substantial increases in erythropoietic output. We also show that combining the expression of the cDNA of a truncated erythropoietin receptor with a previously reported genome-editing strategy to fully replace the HBA1 gene with an HBB transgene in HSPCs (to restore normal haemoglobin production in cells with a ß-thalassaemia phenotype) gives the edited HSPCs and the healthy red blood cell phenotype a proliferative advantage. Combining knowledge of human genetics with precise genome editing to insert natural human variants into therapeutic cells may facilitate safer and more effective genome-editing therapies for patients with genetic diseases.

Secreted Particle Information Transfer (SPIT) - A Cellular Platform for In Vivo Genetic Engineering.

A multitude of tools now exist that allow us to precisely manipulate the human genome in a myriad of different ways. However, successful delivery of these tools to the cells of human patients remains a major barrier to their clinical implementation. Here we introduce a new cellular approach for in vivo genetic engineering, Secreted Particle Information Transfer (SPIT) that utilizes human cells as delivery vectors for in vivo genetic engineering. We demonstrate the application of SPIT for cell-cell delivery of Cre recombinase and CRISPR-Cas9 enzymes, we show that genetic logic can be incorporated into SPIT and present the first demonstration of human cells as a delivery platform for in vivo genetic engineering in immunocompetent mice. We successfully applied SPIT to genetically modify multiple organs and tissue stem cells in vivo including the liver, spleen, intestines, peripheral blood, and bone marrow. We anticipate that by harnessing the large packaging capacity of a human cell's nucleus, the ability of human cells to engraft into patients' long term and the capacity of human cells for complex genetic programming, that SPIT will become a paradigm shifting approach for in vivo genetic engineering.

Demographic and socioeconomic disparities in receipt of ophthalmology consultation for facial trauma.

OBJECTIVE: Functional outcomes following facial and ocular trauma are time-sensitive and require prompt evaluation to minimise long-term vision loss, yet few studies have systematically evaluated disparities in the management of these cases. This study investigates whether a patient's race/ethnicity, primary language, insurance status, gender or age affects receipt of ophthalmology consultation for facial trauma. METHODS AND ANALYSIS: This study was a retrospective cohort analysis of patients from the Elmhurst City Hospital Trauma Registry in Queens, New York who were seen for facial trauma including open globe injuries and orbital fractures between January 2014 and May 2016. RESULTS: Of the 264 patients included, 43% reported as Hispanic, 23% white, 11% Asian, 8% black and 15% other/unknown. After controlling for confounding variables by multivariable logistic regression, neither race/ethnicity, gender, nor primary language were significantly associated with the likelihood of receiving an ophthalmology consult. However, patients with private insurance had 2.57 times greater odds of receiving an ophthalmology consultation than those with Medicaid or state corrections insurance (95% CI 1.37 to 4.95). As age increased, the likelihood of receiving an ophthalmology consultation decreased (p=0.009); patients 60 years of age and older had one-third the odds of ophthalmology consultation as younger patients (OR 0.33; 95% CI 0.16 to 0.68). CONCLUSIONS: This study highlights that lack of ophthalmology consultation in patients with facial trauma is linked to age and underinsurance. Extra attention must be paid during primary assessments to ensure elderly patients and those with public insurance have equitable access to timely and appropriate care for facial trauma.

Clinically relevant gene editing in hematopoietic stem cells for the treatment of pyruvate kinase deficiency.

Pyruvate kinase deficiency (PKD), an autosomal-recessive disorder, is the main cause of chronic non-spherocytic hemolytic anemia. PKD is caused by mutations in the pyruvate kinase, liver and red blood cell (P KLR) gene, which encodes for the erythroid pyruvate kinase protein (RPK). RPK is implicated in the last step of anaerobic glycolysis in red blood cells (RBCs), responsible for the maintenance of normal erythrocyte ATP levels. The only curative treatment for PKD is allogeneic hematopoietic stem and progenitor cell (HSPC) transplant, associated with a significant morbidity and mortality, especially relevant in PKD patients. Here, we address the correction of PKD through precise gene editing at the PKLR endogenous locus to keep the tight regulation of RPK enzyme during erythropoiesis. We combined CRISPR-Cas9 system and donor recombinant adeno-associated vector (rAAV) delivery to build an efficient, safe, and clinically applicable system to knock in therapeutic sequences at the translation start site of the RPK isoform in human hematopoietic progenitors. Edited human hematopoietic progenitors efficiently reconstituted human hematopoiesis in primary and secondary immunodeficient mice. Erythroid cells derived from edited PKD-HSPCs recovered normal ATP levels, demonstrating the restoration of RPK function in PKD erythropoiesis after gene editing. Our gene-editing strategy may represent a lifelong therapy to correct RPK functionality in RBCs for PKD patients.

Development of ß-globin gene correction in human hematopoietic stem cells as a potential durable treatment for sickle cell disease.

Sickle cell disease (SCD) is the most common serious monogenic disease with 300,000 births annually worldwide. SCD is an autosomal recessive disease resulting from a single point mutation in codon six of the ß-globin gene (HBB). Ex vivo ß-globin gene correction in autologous patient-derived hematopoietic stem and progenitor cells (HSPCs) may potentially provide a curative treatment for SCD. We previously developed a CRISPR-Cas9 gene targeting strategy that uses high-fidelity Cas9 precomplexed with chemically modified guide RNAs to induce recombinant adeno-associated virus serotype 6 (rAAV6)-mediated HBB gene correction of the SCD-causing mutation in HSPCs. Here, we demonstrate the preclinical feasibility, efficacy, and toxicology of HBB gene correction in plerixafor-mobilized CD34+ cells from healthy and SCD patient donors (gcHBB-SCD). We achieved up to 60% HBB allelic correction in clinical-scale gcHBB-SCD manufacturing. After transplant into immunodeficient NSG mice, 20% gene correction was achieved with multilineage engraftment. The long-term safety, tumorigenicity, and toxicology study demonstrated no evidence of abnormal hematopoiesis, genotoxicity, or tumorigenicity from the engrafted gcHBB-SCD drug product. Together, these preclinical data support the safety, efficacy, and reproducibility of this gene correction strategy for initiation of a phase 1/2 clinical trial in patients with SCD.

Gene replacement of α-globin with ß-globin restores hemoglobin balance in ß-thalassemia-derived hematopoietic stem and progenitor cells.

ß-Thalassemia pathology is due not only to loss of ß-globin (HBB), but also to erythrotoxic accumulation and aggregation of the ß-globin-binding partner, α-globin (HBA1/2). Here we describe a Cas9/AAV6-mediated genome editing strategy that can replace the entire HBA1 gene with a full-length HBB transgene in ß-thalassemia-derived hematopoietic stem and progenitor cells (HSPCs), which is sufficient to normalize ß-globin:α-globin messenger RNA and protein ratios and restore functional adult hemoglobin tetramers in patient-derived red blood cells. Edited HSPCs were capable of long-term and bilineage hematopoietic reconstitution in mice, establishing proof of concept for replacement of HBA1 with HBB as a novel therapeutic strategy for curing ß-thalassemia.

The TRACE-Seq method tracks recombination alleles and identifies clonal reconstitution dynamics of gene targeted human hematopoietic stem cells.

Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may enable the treatment of genetic diseases of the blood and immune system. It is now possible to correct mutations at high frequencies in HSPCs by combining CRISPR/Cas9 with homologous DNA donors. Because of the precision of gene correction, these approaches preclude clonal tracking of gene-targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a methodology that utilizes barcoded AAV6 donor template libraries, carrying in-frame silent mutations or semi-randomized nucleotides outside the coding region, to track the in vivo lineage contribution of gene-targeted HSPC clones. By targeting the HBB gene with an AAV6 donor template library consisting of ~20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoid-skewed, and balanced multi-lineage repopulating human HSPC clones in mice. We anticipate this methodology could potentially be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings.

Cas9-AAV6 gene correction of beta-globin in autologous HSCs improves sickle cell disease erythropoiesis in mice.

CRISPR/Cas9-mediated beta-globin (HBB) gene correction of sickle cell disease (SCD) patient-derived hematopoietic stem cells (HSCs) in combination with autologous transplantation represents a recent paradigm in gene therapy. Although several Cas9-based HBB-correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated previously. Here, we use a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated HBB-correction in functional HSCs within the context of autologous transplantation. We discover that long-term multipotent HSCs can be gene corrected ex vivo and stable hemoglobin-A production can be achieved in vivo from HBB-corrected HSCs following autologous transplantation. We observe a direct correlation between increased HBB-corrected myeloid chimerism and normalized in vivo red blood cell (RBC) features, but even low levels of chimerism resulted in robust hemoglobin-A levels. Moreover, this study offers a platform for gene editing of mouse HSCs for both basic and translational research.

Cas9-AAV6-engineered human mesenchymal stromal cells improved cutaneous wound healing in diabetic mice.

Human mesenchymal stromal cells (hMSCs) are a promising source for engineered cell-based therapies in which genetic engineering could enhance therapeutic efficacy and install novel cellular functions. Here, we describe an optimized Cas9-AAV6-based genome editing tool platform for site-specific mutagenesis and integration of up to more than 3 kilobases of exogenous DNA in the genome of hMSCs derived from the bone marrow, adipose tissue, and umbilical cord blood without altering their ex vivo characteristics. We generate safe harbor-integrated lines of engineered hMSCs and show that engineered luciferase-expressing hMSCs are transiently active in vivo in wound beds of db/db mice. Moreover, we generate PDGF-BB- and VEGFA-hypersecreting hMSC lines as short-term, local wound healing agents with superior therapeutic efficacy over wildtype hMSCs in the diabetic mouse model without replacing resident cells long-term. This study establishes a precise genetic engineering platform for genetic studies of hMSCs and development of engineered hMSC-based therapies.

Highly efficient editing of the ß-globin gene in patient-derived hematopoietic stem and progenitor cells to treat sickle cell disease.

Sickle cell disease (SCD) is a monogenic disorder that affects millions worldwide. Allogeneic hematopoietic stem cell transplantation is the only available cure. Here, we demonstrate the use of CRISPR/Cas9 and a short single-stranded oligonucleotide template to correct the sickle mutation in the ß-globin gene in hematopoietic stem and progenitor cells (HSPCs) from peripheral blood or bone marrow of patients with SCD, with 24.5 ± 7.6% efficiency without selection. Erythrocytes derived from gene-edited cells showed a marked reduction of sickle cells, with the level of normal hemoglobin (HbA) increased to 25.3 ± 13.9%. Gene-corrected SCD HSPCs retained the ability to engraft when transplanted into non-obese diabetic (NOD)-SCID-gamma (NSG) mice with detectable levels of gene correction 16-19 weeks post-transplantation. We show that, by using a high-fidelity SpyCas9 that maintained the same level of on-target gene modification, the off-target effects including chromosomal rearrangements were significantly reduced. Taken together, our results demonstrate efficient gene correction of the sickle mutation in both peripheral blood and bone marrow-derived SCD HSPCs, a significant reduction in sickling of red blood cells, engraftment of gene-edited SCD HSPCs in vivo and the importance of reducing off-target effects; all are essential for moving genome editing based SCD treatment into clinical practice.

Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination.

Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for in vitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.

CRISPR/Cas9 Genome Engineering in Engraftable Human Brain-Derived Neural Stem Cells.

Human neural stem cells (NSCs) offer therapeutic potential for neurodegenerative diseases, such as inherited monogenic nervous system disorders, and neural injuries. Gene editing in NSCs (GE-NSCs) could enhance their therapeutic potential. We show that NSCs are amenable to gene targeting at multiple loci using Cas9 mRNA with synthetic chemically modified guide RNAs along with DNA donor templates. Transplantation of GE-NSC into oligodendrocyte mutant shiverer-immunodeficient mice showed that GE-NSCs migrate and differentiate into astrocytes, neurons, and myelin-producing oligodendrocytes, highlighting the fact that GE-NSCs retain their NSC characteristics of self-renewal and site-specific global migration and differentiation. To show the therapeutic potential of GE-NSCs, we generated GALC lysosomal enzyme overexpressing GE-NSCs that are able to cross-correct GALC enzyme activity through the mannose-6-phosphate receptor pathway. These GE-NSCs have the potential to be an investigational cell and gene therapy for a range of neurodegenerative disorders and injuries of the central nervous system, including lysosomal storage disorders.

Identification of preexisting adaptive immunity to Cas9 proteins in humans.

The CRISPR-Cas9 system is a powerful tool for genome editing, which allows the precise modification of specific DNA sequences. Many efforts are underway to use the CRISPR-Cas9 system to therapeutically correct human genetic diseases1-6. The most widely used orthologs of Cas9 are derived from Staphylococcus aureus and Streptococcus pyogenes5,7. Given that these two bacterial species infect the human population at high frequencies8,9, we hypothesized that humans may harbor preexisting adaptive immune responses to the Cas9 orthologs derived from these bacterial species, SaCas9 (S. aureus) and SpCas9 (S. pyogenes). By probing human serum for the presence of anti-Cas9 antibodies using an enzyme-linked immunosorbent assay, we detected antibodies against both SaCas9 and SpCas9 in 78% and 58% of donors, respectively. We also found anti-SaCas9 T cells in 78% and anti-SpCas9 T cells in 67% of donors, which demonstrates a high prevalence of antigen-specific T cells against both orthologs. We confirmed that these T cells were Cas9-specific by demonstrating a Cas9-specific cytokine response following isolation, expansion, and antigen restimulation. Together, these data demonstrate that there are preexisting humoral and cell-mediated adaptive immune responses to Cas9 in humans, a finding that should be taken into account as the CRISPR-Cas9 system moves toward clinical trials.

Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting.

Engineered nuclease-mediated gene targeting through homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) has the potential to treat a variety of genetic hematologic and immunologic disorders. Here, we identify critical parameters to reproducibly achieve high frequencies of RNA-guided (single-guide RNA [sgRNA]; CRISPR)-Cas9 nuclease (Cas9/sgRNA) and rAAV6-mediated HR at the ß-globin (HBB) locus in HSPCs. We identified that by transducing HSPCs with rAAV6 post-electroporation, there was a greater than 2-fold electroporation-aided transduction (EAT) of rAAV6 endocytosis with roughly 70% of the cell population having undergone transduction within 2 hr. When HSPCs are cultured at low densities (1 × 105 cells/mL) prior to HBB targeting, HSPC expansion rates are significantly positively correlated with HR frequencies in vitro as well as in repopulating cells in immunodeficient NSG mice in vivo. We also show that culturing fluorescence-activated cell sorting (FACS)-enriched HBB-targeted HSPCs at low cell densities in the presence of the small molecules, UM171 and SR1, stimulates the expansion of gene-edited HSPCs as measured by higher engraftment levels in immunodeficient mice. This work serves not only as an optimized protocol for genome editing HSPCs at the HBB locus for the treatment of ß-hemoglobinopathies but also as a foundation for editing HSPCs at other loci for both basic and translational research.

A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells.

Translation of the CRISPR-Cas9 system to human therapeutics holds high promise. However, specificity remains a concern especially when modifying stem cell populations. We show that existing rationally engineered Cas9 high-fidelity variants have reduced on-target activity when using the therapeutically relevant ribonucleoprotein (RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, p.R691A, in Cas9 (high-fidelity (HiFi) Cas9) retained the high on-target activity of Cas9 while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically relevant loci (HBB, IL2RG, CCR5, HEXB, and TRAC) in human CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as primary T cells. We also show that HiFi Cas9 mediates high-level correction of the sickle cell disease (SCD)-causing p.E6V mutation in HSPCs derived from patients with SCD. We anticipate that HiFi Cas9 will have wide utility for both basic science and therapeutic genome-editing applications.

Global Transcriptional Response to CRISPR/Cas9-AAV6-Based Genome Editing in CD34+ Hematopoietic Stem and Progenitor Cells.

Genome-editing technologies are currently being translated to the clinic. However, cellular effects of the editing machinery have yet to be fully elucidated. Here, we performed global microarray-based gene expression measurements on human CD34+ hematopoietic stem and progenitor cells that underwent editing. We probed effects of the entire editing process as well as each component individually, including electroporation, Cas9 (mRNA or protein) with chemically modified sgRNA, and AAV6 transduction. We identified differentially expressed genes relative to control treatments, which displayed enrichment for particular biological processes. All editing machinery components elicited immune, stress, and apoptotic responses. Cas9 mRNA invoked the greatest amount of transcriptional change, eliciting a distinct viral response and global transcriptional downregulation, particularly of metabolic and cell cycle processes. Electroporation also induced significant transcriptional change, with notable downregulation of metabolic processes. Surprisingly, AAV6 evoked no detectable viral response. We also found Cas9/sgRNA ribonucleoprotein treatment to be well tolerated, in spite of eliciting a DNA damage signature. Overall, this data establishes a benchmark for cellular tolerance of CRISPR/Cas9-AAV6-based genome editing, ensuring that the clinical protocol is as safe and efficient as possible.

CRISPR/Cas9 ß-globin gene targeting in human haematopoietic stem cells.

The ß-haemoglobinopathies, such as sickle cell disease and ß-thalassaemia, are caused by mutations in the ß-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure ß-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult ß-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for ß-haemoglobinopathies.