Megan D. Hoban

Correction of the sickle-cell disease mutation in human hematopoietic stem/progenitor cells

Sickle cell disease (SCD) is characterized by a single point mutation in the seventh codon of the β-globin gene. Site-specific correction of the sickle mutation in hematopoietic stem cells would allow for permanent production of normal red blood cells. Using zinc-finger nucleases (ZFNs) designed to flank the sickle mutation, we demonstrate efficient targeted cleavage at the β-globin locus with minimal off-target modification. By co-delivering a homologous donor template (either an integrase-defective lentiviral vector or a DNA oligonucleotide), high levels of gene modification were achieved in CD34(+) hematopoietic stem and progenitor cells. Modified cells maintained their ability to engraft NOD/SCID/IL2rγ(null) mice and to produce cells from multiple lineages, although with a reduction in the modification levels relative to the in vitro samples. Importantly, ZFN-driven gene correction in CD34(+) cells from the bone marrow of patients with SCD resulted in the production of wild-type hemoglobin tetramers.

CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells

Targeted genome editing technology can correct the sickle cell disease mutation of the β-globin gene in hematopoietic stem cells. This correction supports production of red blood cells that synthesize normal hemoglobin proteins. Here, we demonstrate that Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system can target DNA sequences around the sickle-cell mutation in the β-globin gene for site-specific cleavage and facilitate precise correction when a homologous donor template is codelivered. Several pairs of TALENs and multiple CRISPR guide RNAs were evaluated for both on-target and off-target cleavage rates. Delivery of the CRISPR/Cas9 components to CD34+ cells led to over 18% gene modification in vitro. Additionally, we demonstrate the correction of the sickle cell disease mutation in bone marrow derived CD34+ hematopoietic stem and progenitor cells from sickle cell disease patients, leading to the production of wild-type hemoglobin. These results demonstrate correction of the sickle mutation in patient-derived CD34+ cells using CRISPR/Cas9 technology.

Dissecting the Mechanism of Histone Deacetylase Inhibitors to Enhance the Activity of Zinc Finger Nucleases Delivered by Integrase-Defective Lentiviral Vectors

Integrase-defective lentiviral vectors (IDLVs) have been of limited success in the delivery of zinc finger nucleases (ZFNs) to human cells, due to low expression. A reason for reduced gene expression has been proposed to involve the epigenetic silencing of vector genomes, carried out primarily by histone deacetylases (HDACs). In this study, we tested valproic acid (VPA), a known HDAC inhibitor (HDACi), for its ability to increase transgene expression from IDLVs, especially in the context of ZFN delivery. Using ZFNs targeting the human adenosine deaminase (ADA) gene in K562 cells, we demonstrated that treatment with VPA enhanced ZFN expression by up to 3-fold, resulting in improved allelic disruption at the ADA locus. Furthermore, three other U.S. Food and Drug Administration-approved HDACis (vorinostat, givinostat, and trichostatin-A) exhibited a similar effect on the activity of ZFN-IDLVs in K562 cells. In primary human CD34(+) cells, VPA- and vorinostat-treated cells showed higher levels of expression of both green fluorescent protein (GFP) as well as ZFNs from IDLVs. A major mechanism for the effects of HDAC inhibitors on improving expression was from their modulation of the cell cycle, and the influence of heterochromatinization was determined to be a lesser contributing factor.

The human ankyrin 1 promoter insulator sustains gene expression in a β-globin lentiviral vector in hematopoietic stem cells.

Lentiviral vectors designed for the treatment of the hemoglobinopathies require the inclusion of regulatory and strong enhancer elements to achieve sufficient expression of the β-globin transgene. Despite the inclusion of these elements, the efficacy of these vectors may be limited by transgene silencing due to the genomic environment surrounding the integration site. Barrier insulators can be used to give more consistent expression and resist silencing even with lower vector copies. Here, the barrier activity of an insulator element from the human ankyrin-1 gene was analyzed in a lentiviral vector carrying an antisickling human β-globin gene. Inclusion of a single copy of the Ankyrin insulator did not affect viral titer, and improved the consistency of expression from the vector in murine erythroleukemia cells. The presence of the Ankyrin insulator element did not change transgene expression in human hematopoietic cells in short-term erythroid culture or in vivo in primary murine transplants. However, analysis in secondary recipients showed that the lentiviral vector with the Ankyrin element preserved transgene expression, whereas expression from the vector lacking the Ankyrin insulator decreased in secondary recipients. These studies demonstrate that the Ankyrin insulator may improve long-term β-globin expression in hematopoietic stem cells for gene therapy of hemoglobinopathies.

Delivery of Genome Editing Reagents to Hematopoietic Stem/Progenitor Cells.

This unit describes the protocol for the delivery of reagents for targeted genome editing to CD34(+) hematopoietic stem/progenitor cells (HSPCs). Specifically, this unit focuses on the process of thawing and pre-stimulating CD34(+) HSPCs, as well as the details of their electroporation with in vitro-transcribed mRNA-encoding site-specific nucleases [in this case zinc-finger nucleases (ZFNs)]. In addition, discussed is delivery of a gene editing donor template in the form of an oligonucleotide or integrase-defective lentiviral vector (IDLV). Finally, an analysis of cell survival following treatment and downstream culture conditions are presented. While optimization steps might be needed for each specific application with respect to nuclease and donor template amount, adherence to this protocol will serve as an excellent starting point for this further work.

Characterization of Gene Alterations following Editing of the β-Globin Gene Locus in Hematopoietic Stem/Progenitor Cells

The use of engineered nucleases combined with a homologous DNA donor template can result in targeted gene correction of the sickle cell disease mutation in hematopoietic stem and progenitor cells. However, because of the high homology between the adjacent human β- and δ-globin genes, off-target cleavage is observed at δ-globin when using some endonucleases targeted to the sickle mutation in β-globin. Introduction of multiple double-stranded breaks by endonucleases has the potential to induce intergenic alterations. Using a novel droplet digital PCR assay and high-throughput sequencing, we characterized the frequency of rearrangements between the β- and δ-globin paralogs when delivering these nucleases. Pooled CD34+ cells and colony-forming units from sickle bone marrow were treated with nuclease only or including a donor template and then analyzed for potential gene rearrangements. It was observed that, in pooled CD34+ cells and colony-forming units, the intergenic β-δ-globin deletion was the most frequent rearrangement, followed by inversion of the intergenic fragment, with the inter-chromosomal translocation as the least frequent. No rearrangements were observed when endonuclease activity was restricted to on-target β-globin cleavage. These findings demonstrate the need to develop site-specific endonucleases with high specificity to avoid unwanted gene alterations.

118. Characterization of Chromosomal Alterations Using a Zinc-Finger Nuclease Targeting the Beta-Globin Gene Locus in Hematopoietic Stem/Progenitor Cells

The use of engineered nucleases for targeted gene correction of the sickle cell disease (SCD) mutation in hematopoietic stem and progenitor cells (HSPCs) combined with a homologous DNA donor template can result in successful targeted gene correction. However, due to a high sequence homology existing between the beta- and delta-globin genes, off-target endonuclease cleavage events can occur when using endonucleases targeted to beta-globin. Moreover, the introduction of multiple double stranded breaks by site-specific endonucleases has the potential to induce chromosomal alterations such as translocations, deletions, and inversions. This work focused on the use of a novel Droplet Digital PCR (ddPCR) assay to characterize the frequency of deletions, inversions and translocations between the beta- and delta-globin paralogs when delivering nucleases that can cleave at beta- and show off-target activity at the delta-globin locus in HSPCs. The impact of co-delivering these nucleases with or without a homology-directed repair (HDR) donor template (as an integrase defective lentiviral vector [IDLV] or as a single stranded oligonucleotide) was also assessed. Bone marrow (BM) CD34+ cells from a SCD patient were treated with ZFNs with high levels of site-specific cleavage activity at both the beta- and delta-globin loci (32-44% indels at beta-globin and 2-12% indels at delta-globin by CEL1 assay). Samples treated with nuclease-only, nuclease+Oligo and nuclease+IDLV respectively, showed that the different events were present at the following frequencies: 41.3%, 45.3% and 50.2% for the deletion; 9.7%, 6.5% and 8.8% for the inversion; and 0.6%, 0.5% and 0.2% for the translocations involving beta- and delta-globin. The same analysis performed in colony forming units (CFUs), derived from the SCD BM CD34+ cells treated with nuclease or nuclease with HDR Donor, showed that the deletion was the most frequent event, followed by the inversion, with the translocation least frequent, mirroring what was observed in the CD34+ pooled populations. Thus, for each event, the frequency was independent of the presence of oligo or IDLV donor template, in both the pooled and the clonal populations. Additionally, the ddPCR analysis of CFUs gave insight into the mono-allelic vs. bi-allelic nature of each rearrangement event, and thus the mono-allelic vs. bi-allelic modification frequency of the nuclease-driven editing. Analysis of the nuclease only treated CFUs showed that 30% of deletion events were bi-allelic; while the majority of inversions and translocations were mono-allelic. These results confirmed that due to the high sequence homology existing between the target locus in beta-globin and the off-target locus in delta-globin, there was a significant frequency of chromosomal alterations induced by cleaving both paralogs in the same human HSPCs. This may partly reflect the unique chromosomal conformation of the globin genes due to their co-regulation in hematopoiesis, and looping to the same cis-regulatory element. More generally, these findings demonstrate the need to develop site-specific endonucleases with high specificity to avoid unwanted chromosomal alterations.

121. Targeted Gene Therapy in the Treatment of X-Linked Hyper IgM Syndrome

X-linked hyper-IgM syndrome results in absent IgG, IgA, IgE and normal/elevated IgM due to defects in the CD40 ligand gene. HSCT is the only curative modality, but it carries significant risks, suggesting the need for improved methods of treatment. TALENs or CRISPRs, combined with the effective delivery of a homologous donor sequence containing normal CD40L DNA, will allow for targeted integration and provide physiologic expression of the endogenous CD40L gene to provide long-term immune reconstitution. TALENs targeting the 5′ UTR of the CD40L gene lead to allelic disruption of up to 31%at the target locus inK562 cells. In order to evaluate the capacity for targeted integration of a cassette at the cut site, cells were electroporated with the TALEN pair and a donor molecule with a promoterless GFP reporter gene flanked by homology arms. Expression of the GFP reporter was evaluated in Jurkat cells, with up to 10 % detected by flow cytometry and increasing to 22 % upon PHA activation. In addition, CRISPRs targeting a patient-specific mutation in intron 3 achieved >50 % gene disruption in K562 cells. Co-electroporation with a donormodified to contain a unique restriction enzyme site demonstrated site-specific gene integration. Site-specific modification at CD40L is achievable and physiologic expression of the endogenous CD40L gene could provide a viable therapy for XHIM.

338. Evaluation of TALENs and the CRISPR/Cas9 Nuclease System To Correct the Sickle Cell Disease Mutation

Targeted genome editing technology can correct the sickle cell disease mutation of the beta-globin gene in hematopoietic stem and progenitor cells (HSCs). The correction induces production of red blood cells that synthesize normal hemoglobin proteins. Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease systems have been developed to target the sickle mutation in the beta-globin gene for site-specific cleavage to facilitate precise correction of the sickle mutation by a co-delivered homologous donor template. K562 cells were electroporated with TALEN and CRISPR/Cas9 expression plasmids and, using the Surveyor Nuclease Assay (Cel-1), cleavage rates were quantified and compared between TALEN- and CRISPR/Cas9-treated cells. Of the six CRISPR/Cas9 guides tested, each of them led to target disruption of the beta-globin locus with the highest cleavage rates upwards of 35% of alleles. Of 4 distinct TALEN pairs generated, only 2 demonstrated targeted cleavage at rates nearing 10% of alleles. In addition to on-target cleavage at beta-globin, nuclease off-target cleavage at other beta-globin family genes was evaluated for each technology by Cel-1 of nucleofected K562 cells. Here the two TALEN pairs demonstrate cleavage in the highly-homologous delta-globin gene with the optimal TALEN pair cleaving 11% of alleles. In this assay, of the 6 CRISPR guides tested, none showed off-target disruption of delta-globin or any of the other beta-globin cluster genes. Of note, in each of the tested guides, at least one base differed from the target site in beta-globin to the respective sequence in delta-globin in the 10bp PAM proximal region. Further experiments are being conducted to determine the genome-wide off-target effects of each of these nucleases.Upon co-delivery of a plasmid donor template containing the corrective base at the sickle site as well as a restriction fragment length polymorphism (RFLP) for rapid assessment of targeted gene modification, both nuclease technologies led to gene modification. Gene modification rates were assayed by qPCR with primers specific to the modified base. TALENs drove gene modification rates of 18%, while the optimal CRISPR guides resulted in 37% modification in K562 cells without sorting for transfected cells. These results provide the basis for pursuing the use of the CRISPR/Cas9 nuclease system for targeted correction of the sickle mutation in human HSCs.