A Novel RPS19-Edited Hematopoietic Stem Cell Model of Diamond-Blackfan Anemia for Development of Lentiviral Vector Gene Therapy

Diamond-Blackfan anemia (DBA) is a congenital hypoplastic anemia that typically manifests in infancy as macrocytic anemia with reticulocytopenia. About 80% of DBA cases are caused by heterozygous loss-of-function mutations or deletions in one of 23 ribosomal proteins (RP) genes, with RPS19 being affected in ~25% of patients. Current therapies are suboptimal, and it is difficult to obtain DBA patient hematopoietic stem and progenitor cells (HSPCs) in sufficient quantity for preclinical development of new therapies. To address this gap, we used CRISPR/Cas9-edited healthy donor CD34 + HSPCs to create a novel model of RPS19-mutated DBA and used this model to develop an efficient RPS19-encoding lentiviral vector (LV) for gene therapy. Healthy donor CD34 + HSPCs were electroporated with ribonucleoprotein (RNP) complex consisting of Cas9 and guide RNAs (gRNAs) targeting RPS19 or the AAVS1 locus as a negative control, then grown in medium to support erythroid differentiation. All gRNAs analyzed generated high-frequency on-target insertion-deletion (indels) mutations (Fig. A). RPS19 indels specifically declined over time, suggesting that RPS19 disruption impairs cell proliferation and/or survival. To rescue the defect, we constructed a third-generation, self-inactivating LV expressing RPS19+GFP (RPS19/GFP LV). Transduction was optimized using poloxamer and prostaglandin E2 and linear transduction efficiency was noted even at high multiplicity of infection (MOI) (Fig. B). An MOI of 20 was used for subsequent experiments. In methylcellulose medium, RPS19 RNP-treated cells generated 72% fewer burst forming unit-erythroid (BFU-E) colonies compared to AAVS1 RNP-treated control cells. RPS19/GFP LVs with three different promoters (EF1α short, EF1α long and MND) partially restored BFU-E formation similarly (Fig. C); the EF1α short promoter was chosen for subsequent experiments due its track record for clinical use. We down-titrated the RNP concentration to generate a total indel frequency of ~25%, which resulted in approximately equal frequencies of RPS19 +/+ and RPS19 +/- BFU-E colonies. RPS19 -/- colonies were detected only after edited HSPCs were rescued by RPS19/GFP LV, due to lethality of this genotype (Fig. D). Transfection of CD34 + HSPCs with RPS19 RNP caused a 49% reduction in cell number after 14 days of liquid culture in erythroid differentiation medium compared to control HSPCs; this was corrected by treatment with RPS19/GFP LV (Fig. E). RPS19 RNP treatment of CD34 + HSPCs had no effect on the expansion of cells grown under myeloid differentiation conditions (Fig. F). We analyzed RNP-treated CD34 + cells further by transducing them with RPS19/GFP LV or control LV encoding GFPalone, transplanting them into immunodeficient NSGW mice and analyzing human donor cell progeny in mouse bone marrow after 16 weeks. In cells treated with AAVS1 RNP and GFP LV, the indel frequency dropped from 27.2±1.5% (SD) at 72 hours after editing (input) to 15.5±4.4% at 16 weeks post-transplant (43% reduction) (Fig. G). In HSPCs treated with RPS19 RNP and GFP LV the indel frequency dropped from 20.9±3.1% in input cells to 1.8±0.9% after 16 weeks (Fig. G) (92% reduction). In contrast, the indel frequency of donor HSPCs treated with RPS19 RNP and RPS19/GFP LV dropped from 23.6±2.7% in input cells to 8.4±1.6% (64% reduction), which represents a 5-fold increase in indel frequency compared to treatment with control GFP LV (p< 0.01)(Fig. G). In flow cytometry-purified, donor HSPC-derived myeloid, B-lymphocyte, HSPC and erythroid lineages at 16 weeks after xenotransplantation, the mean indel rates were 1.3% to 2.5% in cells derived from HSPCs treated with RPS19 RNP and GFP LV. Indel rates ranged from 6.9% to 9.2% in the progeny of input HSPCs that were rescued by RPS19/GFP LV, representing a 4-6-fold increase compared to transduction with control GFP LV (p<0.01) (Fig. H). In summary, our studies show that Cas9-mediated disruption of RPS19 in CD34 + HSPCs causes a selective erythroid defect in RPS19 +/- cells, recapitulating the canonical DBA defect. Additionally, deficient bone marrow repopulation by RPS19 +/- cells suggests an HSC defect, consistent with pancytopenia that is observed in many older DBA patients. The optimized RPS19 LV transduces HSPCs at high efficiency and alleviates both defects, supporting its potential utility for DBA therapy. Figure 1 Figure 1. Disclosures Yen: Beam Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Weiss: Beam Therapeutics: Current holder of stock options in a privately-held company; Forma Therapeutics: Consultancy; Novartis: Consultancy; Cellarity Inc.: Consultancy.

Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs delivered by adeno-associated virus

Gene therapy would benefit from a miniature CRISPR system that fits into the small adeno-associated virus (AAV) genome and has high cleavage activity and specificity in eukaryotic cells. One of the most compact CRISPR-associated nucleases yet discovered is the archaeal Un1Cas12f1. However, Un1Cas12f1 and its variants have very low activity in eukaryotic cells. In the present study, we redesigned the natural guide RNA of Un1Cas12f1 at five sites: the 5′ terminus of the trans-activating CRISPR RNA (tracrRNA), the tracrRNA–crRNA complementary region, a penta(uridinylate) sequence, the 3′ terminus of the crRNA and a disordered stem 2 region in the tracrRNA. These optimizations synergistically increased the average indel frequency by 867-fold. The optimized Un1Cas12f1 system enabled efficient, specific genome editing in human cells when delivered by plasmid vectors, PCR amplicons and AAV. As Un1Cas12f1 cleaves outside the protospacer, it can be used to create large deletions efficiently. The engineered Un1Cas12f1 system showed efficiency comparable to that of SpCas9 and specificity similar to that of AsCas12a.

Reversible gene silencing through frameshift indels and frameshift scars provide adaptive plasticity for Mycobacterium tuberculosis

Mycobacterium tuberculosis can adapt to changing environments by non-heritable mechanisms. Frame-shifting insertions and deletions (indels) may also participate in adaptation through gene disruption, which could be reversed by secondary introduction of a frame-restoring indel. We present ScarTrek, a program that scans genomic data for indels, including those that together disrupt and restore a gene’s reading frame, producing “frame-shift scars” suggestive of reversible gene inactivation. We use ScarTrek to analyze 5977 clinical M. tuberculosis isolates. We show that indel frequency inversely correlates with genomic linguistic complexity and varies with gene-position and gene-essentiality. Using ScarTrek, we detect 74 unique frame-shift scars in 48 genes, with a 3.74% population-level incidence of unique scar events. We find multiple scars in the ESX-1 gene cluster. Six scars show evidence of convergent evolution while the rest shared a common ancestor. Our results suggest that sequential indels are a mechanism for reversible gene silencing and adaptation in M. tuberculosis.

TSA Promotes CRISPR/Cas9 Editing Efficiency and Expression of Cell Division-Related Genes from Plant Protoplasts

Trichostatin A (TSA) is a representative histone deacetylase (HDAC) inhibitor that modulates epigenetic gene expression by regulation of chromatin remodeling in cells. To investigate whether the regulation of chromatin de-condensation by TSA can affect the increase in the efficiency of Cas9 protein-gRNA ribonucleoprotein (RNP) indel formation from plant cells, genome editing efficiency using lettuce and tobacco protoplasts was examined after several concentrations of TSA treatments (0, 0.1, 1 and 10 μM). RNP delivery from protoplasts was conducted by conventional polyethylene glycol (PEG) transfection protocols. Interestingly, the indel frequency of the SOC1 gene from TSA treatments was about 3.3 to 3.8 times higher than DMSO treatment in lettuce protoplasts. The TSA-mediated increase of indel frequency of the SOC1 gene in lettuce protoplasts occurred in a concentration-dependent manner, although there was not much difference. Similar to lettuce, TSA also increased the indel frequency by 1.5 to 1.8 times in a concentration-dependent manner during PDS genome editing using tobacco protoplasts. The MNase test clearly showed that chromatin accessibility with TSA treatments was higher than that of DMSO treatment. Additionally, TSA treatment significantly increased the level of histone H3 and H4 acetylation from lettuce protoplasts. The qRT-PCR analysis showed that expression of cell division-related genes (LsCYCD1-1, LsCYCD3-2, LsCYCD6-1, and LsCYCU4-1) was increased by TSA treatment. These findings could contribute to increasing the efficiency of CRISPR/Cas9-mediated genome editing. Furthermore, this could be applied for the development of useful genome-edited crops using the CRISPR/Cas9 system with plant protoplasts.

Gene Editing ELANE in Human Hematopoietic Stem and Progenitor Cells Reveals Disease Mechanisms and Therapeutic Strategies for Severe Congenital Neutropenia

Severe congenital neutropenia (SCN) is a life-threatening disorder of insufficient granulocytes. Lifelong granulocyte colony-stimulating factor (G-CSF) injections are the mainstay of treatment, yet there remains a high risk of myelodysplastic syndrome and acute myeloid leukemia. The most common etiology of SCN is germline ELANE mutation. These dominantly acting mutations preserve expression but alter the structure of the neutrophil elastase protein product resulting in altered protein folding and/or trafficking with excess cell death at the promyelocyte/myelocyte stage of maturation. Recent advances in gene editing technologies have enabled targeted genetic modification of hematopoietic stem cells (HSCs); nonetheless genetic repair of specific disease-associated mutations remains challenging. We hypothesized that introduction of premature termination codons (PTCs) by nuclease-mediated frameshift mutations within early exons of ELANE could constitute a universal, highly efficient, simple therapeutic approach for ELANE-associated SCN. We predicted that the PTCs would trigger nonsense mediated decay (NMD) of the mutant transcript resulting in its loss of expression and thus bypassing neutrophil precursor cell death and consequent neutropenia. The mild phenotype observed in the Papillon-Lefevre syndrome, characterized by combined serine protease deficiency, suggests that isolated neutrophil elastase deficiency would not result in clinically significant immunodeficiency. We delivered 3xNLS-SpCas9 and ELANE targeting sgRNA as ribonucleoprotein (RNP) complexes to primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) and conducted in vitro neutrophil maturation culture. Introducing indels at exon 2 of ELANE efficiently triggered NMD. Edited cells were fully competent for neutrophil maturation similar to neutral locus targeted control cells. Using three human donors, we found that ELANE exon 2 edited HSPCs produced similar human bone marrow (BM) chimerism as unedited cells in NBSGW recipient mice 16 weeks following infusion. We found similar lymphoid, erythroid, and myeloid engraftment including similar fraction of human neutrophils (13.4% of total human BM cells in unedited and 13.7% in ELANE exon 2 edited, despite 97.3% on-target indel frequency and 84.3% reduction in ELANE expression in the latter). Using CD34+ HSPCs from four ELANE mutant SCN patient donors, we demonstrated that exon 2 targeting RNPs achieve highly efficient editing exceeding 95% indel frequency, trigger ELANE transcript decay, and rescue promyelocyte stage maturation arrest. In contrast to these ameliorating early exon frameshifts, naturally occurring SCN-associated frameshifts affect late exons of ELANE, suggesting that these mutations might escape NMD. Indeed we found that targeting ELANE exon 5 in HSPCs resulted in robust indels (93.5%), preserving ELANE expression but resulting in cell death at the promyelocyte/myelocyte stages of development, recapitulating an SCN phenotype. To our surprise, we found that only -1 frameshifts and not -2 frameshifts induced by gene editing with NHEJ repair led to the SCN-like phenotype, although we noted that all 23 reported naturally occurring SCN-associated ELANE frameshift mutations result from -1 but not -2 bp frameshifts. Using xenograft of NBSGW recipients, we found that an RNP complex leading to efficient -1 frame indels in ELANE exon 5 produced profound neutrophil maturation block, with reduction from 13.4% neutrophils in controls to 0.5% neutrophils in ELANE exon 5 targeted recipients, with otherwise indistinguishable human monocyte, lymphoid, and erythroid reconstitution as compared to controls. This dramatic phenotype contrasts with mice engineered to express SCN-associated Elane mutations that do not exhibit neutropenia, indicating species differences in granulopoiesis. Together these results support the development of ELANE early exon targeting as a highly efficient universal therapy for ELANE mutant SCN, feasible with existing gene editing technology. Moreover, by late exon ELANE gene editing we have developed a robust new model of SCN using primary human HSPCs that recapitulates neutropenia in vivo following xenotransplant, refines the molecular genetics of mutant ELANE induced neutrophil maturation arrest, and offers opportunities to explore novel therapeutic approaches. Disclosures Newburger: TransCytos LLC: Consultancy; X4 Pharmaceuticals: Consultancy, Honoraria.

An Engineered Cell-Traceable Model of Reticular Dysgenesis in Human Hematopoietic Stem Cells Linking Metabolism and Differentiation

Hematopoietic stem cell (HSC) differentiation is accompanied by a metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS) to meet the increasing energy demand during proliferation and differentiation. However, the role of mitochondrial metabolism in HSC differentiation goes beyond ATP production. Metabolites generated during mitochondrial metabolism may impact in HSC fate decisions through stable epigenetic modifications. Despite some progress in understanding mitochondrial communication during HSC development, their role in human hematopoiesis remains largely elusive, where the lack of appropriate model systems poses a major obstacle. Reticular Dysgenesis (RD), a rare and particularly severe form of severe combined immunodeficiency (SCID), offers an attractive model for studying the role of mitochondrial metabolism in hematopoiesis. RD is an autosomal recessive disease caused by biallelic mutations of the mitochondrial enzyme Adenylate Kinase 2 (AK2). AK2 catalyzes the reversible phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP), which serves as the substrate for the ATP synthase. In addition to defective lymphocyte development typical of classic SCID, RD patients also suffer from impaired myeloid development, suggestive of a global defect in hematopoiesis. In a human induced pluripotent stem cell (iPSC) model for RD, hematopoietic stem and progenitor cells (HSPCs) recapitulate a profound maturation arrest of the myeloid lineage, increased oxidative stress and an energy-depleted metabolite and transcriptional profile. We hypothesize that AK2 defects drive hematopoietic cell fate decisions through changes in metabolites that regulate the activities of DNA/histone modifying enzymes and result in stable epigenetic modifications. Methods: Since iPSCs are not suitable to model the epigenetic characteristics of definitive hematopoiesis, we developed a novel model system in which we deleted AK2 in primary human HSCs using CRISPR/Cas9 gene editing technique. We found a highly effective single-guide RNA (sgRNA) targeting the catalytic LID domain of the AK2 gene to introduce directed DNA double stranded breaks (DSBs), and use a homologous recombination (HR)-mediated dual reporter system to track and isolate cells with biallelic AK2 disruption. Results: Our single-color GFP reporter system consistently produces a >60% GFP+ population of AK2-targeted CD34+ umbilical cord blood (UCB) cells. With dual GFP/BFP reporters, we were able to achieve 6% GFP/BFP double positive cells with confirmed biallelic AK2 knock-out. Since HR events on one allele are biologically linked to CRISPR/Cas9 mediated DSBs on the other, we assessed insertion and/or deletion (INDEL) frequency and protein expression in a single reporter (GFP+) population of AK2-targeted UCBs. We detected an INDEL frequency of over 90% on the non-HR alleles along with nearly absent AK2 protein expression by Western Blot. These results indicated that the highly efficient single-color reporter system with >60% targeting efficiency is sufficient to achieve an AK2 biallelic knock-out population in primary HSCs. in vitro myeloid differentiation of these AK2-targeted HSCs recapitulates the RD phenotype with impaired neutrophil but preserved monocyte development. Conclusion: This novel disease model for RD will now allow us to examine the cellular and molecular impact of perturbations in metabolism on human HSC development. We will investigate the effect on differentiation potential, metabolite profile, transcriptome and epigenome in vitro as well as in a xenograft mouse model. Elucidating how metabolism governs differentiation and self-renewal of human HSCs will not only advance our basic understanding of many blood and immune diseases, but has important translational implications for improving the use of HSCs in hematopoietic stem cell transplantation, gene and cell therapy. Disclosures Porteus: CRISPR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees.

IMPROVING THE PERFORMANCE OF PROTEIN THREADING USING INSERTION/DELETION FREQUENCY ARRAYS

As a protein evolves, not every part of the amino acid sequence has an equal probability of being deleted or for allowing insertions, because not every amino acid plays an equally important role in maintaining the protein structure. However, the most prevalent models in fold recognition methods treat every amino acid deletion and insertion as equally probable events. We have analyzed the alignment patterns for homologous and analogous sequences to determine patterns of insertion and deletion, and used that information to determine the statistics of insertions and deletions for different amino acids of a target sequence. We define these patterns as insertion/deletion (indel) frequency arrays (IFAs). By applying IFAs to the protein threading problem, we have been able to improve the alignment accuracy, especially for proteins with low sequence identity. We have also demonstrated that the application of this information can lead to an improvement in fold recognition.