Ex vivo culture resting time impacts transplantation outcomes of genome-edited human hematopoietic stem and progenitor cells in xenograft mouse models.

Ex vivo resting culture is a standard procedure following genome editing in hematopoietic stem and progenitor cells (HSPCs). However, prolonged culture may critically affect cell viability and stem cell function. We investigated whether varying durations of culture resting times impact the engraftment efficiency of human CD34+ HSPCs edited at the BCL11A enhancer, a key regulator in the expression of fetal hemoglobin. We employed electroporation to introduce CRISPR-Cas9 components for BCL11A enhancer editing and compared outcomes with nonelectroporated (NEP) and electroporated-only (EP) control groups. Post-electroporation, we monitored cell viability, death rates, and the frequency of enriched hematopoietic stem cell (HSC) fractions (CD34+CD90+CD45RA- cells) over a 48-hour period. Our findings reveal that while the NEP group showed an increase in cell numbers 24 hours post-electroporation, both EP and BCL11A-edited groups experienced significant cell loss. Although CD34+ cell frequency remained high in all groups for up to 48 hours post-electroporation, the frequency of the HSC-enriched fraction was significantly lower in the EP and edited groups compared to the NEP group. In NBSGW xenograft mouse models, both conditioned with busulfan and nonconditioned, we found that immediate transplantation post-electroporation led to enhanced engraftment without compromising editing efficiency. Human glycophorin A+ (GPA+) red blood cells (RBCs) sorted from bone marrow of all BCL11A edited mice exhibited similar levels of γ-globin expression, regardless of infusion time. Our findings underscore the critical importance of optimizing the culture duration between genome editing and transplantation. Minimizing this interval may significantly enhance engraftment success and minimize cell loss without compromising editing efficiency. These insights offer a pathway to improve the success rates of genome editing in HSPCs, particularly for conditions like sickle cell disease.

Fertility-preserving myeloablative conditioning using single-dose CD117 antibody-drug conjugate in a rhesus gene therapy model.

Hematopoietic stem cell (HSC) gene therapy has curative potential; however, its use is limited by the morbidity and mortality associated with current chemotherapy-based conditioning. Targeted conditioning using antibody-drug conjugates (ADC) holds promise for reduced toxicity in HSC gene therapy. Here we test the ability of an antibody-drug conjugate targeting CD117 (CD117-ADC) to enable engraftment in a non-human primate lentiviral gene therapy model of hemoglobinopathies. Following single-dose CD117-ADC, a >99% depletion of bone marrow CD34 + CD90 + CD45RA- cells without lymphocyte reduction is observed, which results are not inferior to multi-day myeloablative busulfan conditioning. CD117-ADC, similarly to busulfan, allows efficient engraftment, gene marking, and vector-derived fetal hemoglobin induction. Importantly, ADC treatment is associated with minimal toxicity, and CD117-ADC-conditioned animals maintain fertility. In contrast, busulfan treatment commonly causes severe toxicities and infertility in humans. Thus, the myeloablative capacity of single-dose CD117-ADC is sufficient for efficient engraftment of gene-modified HSCs while preserving fertility and reducing adverse effects related to toxicity in non-human primates. This targeted conditioning approach thus provides the proof-of-principle to improve risk-benefit ratio in a variety of HSC-based gene therapy products in humans.

Pre-existing immunity does not impair the engraftment of CRISPR-Cas9-edited cells in rhesus macaques conditioned with busulfan or radiation.

CRISPR-Cas9-based therapeutic genome editing approaches hold promise to cure a variety of human diseases. Recent findings demonstrate pre-existing immunity for the commonly used Cas orthologs from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) in humans, which threatens the success of this powerful tool in clinical use. Thus, a comprehensive investigation and potential risk assessment are required to exploit the full potential of the system. Here, we investigated existence of immunity to SpCas9 and SaCas9 in control rhesus macaques (Macaca mulatta) alongside monkeys transplanted with either lentiviral transduced or CRISPR-SpCas9 ribonucleoprotein (RNP)-edited cells. We observed significant levels of Cas9 antibodies in the peripheral blood of all transplanted and non-transplanted control animals. Transplantation of ex vivo transduced or SpCas9-mediated BCL11A enhancer-edited cells did not alter the levels of Cas9 antibodies in rhesus monkeys. Following stimulation of peripheral blood cells with SpCas9 or SaCas9, neither Cas9-specific T cells nor cytokine induction were detected. Robust and durable editing frequencies and expression of high levels of fetal hemoglobin in BCL11A enhancer-edited rhesus monkeys with no evidence of an immune response (>3 years) provide an optimistic outlook for the use of ex vivo CRISPR-SpCas9 (RNP)-edited cells.

Self-organized yolk sac-like organoids allow for scalable generation of multipotent hematopoietic progenitor cells from induced pluripotent stem cells.

Although the differentiation of human induced pluripotent stem cells (hiPSCs) into various types of blood cells has been well established, approaches for clinical-scale production of multipotent hematopoietic progenitor cells (HPCs) remain challenging. We found that hiPSCs cocultured with stromal cells as spheroids (hematopoietic spheroids [Hp-spheroids]) can grow in a stirred bioreactor and develop into yolk sac-like organoids without the addition of exogenous factors. Hp-spheroid-induced organoids recapitulated a yolk sac-characteristic cellular complement and structures as well as the functional ability to generate HPCs with lympho-myeloid potential. Moreover, sequential hemato-vascular ontogenesis could also be observed during organoid formation. We demonstrated that organoid-induced HPCs can be differentiated into erythroid cells, macrophages, and T lymphocytes with current maturation protocols. Notably, the Hp-spheroid system can be performed in an autologous and xeno-free manner, thereby improving the feasibility of bulk production of hiPSC-derived HPCs in clinical, therapeutic contexts.

Evaluation of genetic diversity using 31 microsatellites in Miyako horses.

The Miyako horse is a native Japanese horse breed. As with other native Japanese horses, the number of Miyako horses decreased due to mechanization and motorization, which reduced their roles, with just 14 in 1980. Although their population had increased to 55 horses by 2021, a further increase in their numbers is required to avoid extinction. Recently, their breeding has involved natural mating during group grazing; therefore, pedigree management has been difficult, and individual identification has been inconclusive. With the aim of formulating an effective breeding plan, this study used microsatellites to confirm parent-offspring relationships and evaluate the genetic diversity over time. First, the combination of microsatellite genotypes identified misunderstood parent-offspring relationships in 35.3% of the existing individuals, and a correct family tree was reconstructed. Next, the number of alleles and observed and expected values of heterozygosity were calculated separately for the populations during periods of 1998-2012 and 2013-2020. The values were 4.2, 0.705, and 0.653 and 3.9, 0.633, and 0.603, respectively, indicating that genetic diversity according to all indices decreased during period of 2013-2020. This was probably because of the bias of stallions in the 2013-2020 population. Errors in pedigree information in a small population such as Miyako horses could increase the risk of inbreeding, and confirmation of parent-offspring relationships using genotypes may be beneficial. Additionally, to maintain diversity in future breeding, it is important to avoid bias, particularly among stallions, and to ensure offspring of various individuals who are as distantly related to each other as possible.

Large-scale purification of functional AAV particles packaging the full genome using short-term ultracentrifugation with a zonal rotor.

Adeno-associated virus (AAV) vector-based gene therapy is potentially curative for various genetic diseases; however, the development of a scalable purification method for full-genome AAV vectors remains crucial to increase productivity and reduce cost of GMP production. In this study, we developed a large-scale short-term purification method for functional full-genome AAV particles by using 2-step cesium chloride (CsCl) density-gradient ultracentrifugation with a zonal rotor. The 2-step CsCl method with a zonal rotor improves separation between empty and full-genome AAV particles, reducing the ultracentrifugation time (4-5 h) and increasing the AAV volume for purification. The highly purified full-genome AAV particles were confirmed by analytical ultracentrifugation (AUC), droplet digital PCR (ddPCR) in the whole region of the AAV vector genome, transduction efficiency in target cells, and transmission electronic microscopy (TEM). The high-purity AAV9 particles were obtained using culture supernatant during vector preparation rather than cell lysate. CsCl could be simply removed by a hydroxyapatite column. Interestingly, ddPCR analysis revealed that "empty" AAV particles contain small fragments of the inverted terminal repeat (ITR), probably due to unexpected packaging of Rep-mediated ITR fragments. This large-scale functional AAV vector purification with ultracentrifugation would be effective for gene therapy.

Forced enhancer-promoter rewiring to alter gene expression in animal models.

Transcriptional enhancers can be in physical proximity of their target genes via chromatin looping. The enhancer at the ß-globin locus (locus control region [LCR]) contacts the fetal-type (HBG) and adult-type (HBB) ß-globin genes during corresponding developmental stages. We have demonstrated previously that forcing proximity between the LCR and HBG genes in cultured adult-stage erythroid cells can activate HBG transcription. Activation of HBG expression in erythroid cells is of benefit to patients with sickle cell disease. Here, using the ß-globin locus as a model, we provide proof of concept at the organismal level that forced enhancer rewiring might present a strategy to alter gene expression for therapeutic purposes. Hematopoietic stem and progenitor cells (HSPCs) from mice bearing human ß-globin genes were transduced with lentiviral vectors expressing a synthetic transcription factor (ZF-Ldb1) that fosters LCR-HBG contacts. When engrafted into host animals, HSPCs gave rise to adult-type erythroid cells with elevated HBG expression. Vectors containing ZF-Ldb1 were optimized for activity in cultured human and rhesus macaque erythroid cells. Upon transplantation into rhesus macaques, erythroid cells from HSPCs expressing ZF-Ldb1 displayed elevated HBG production. These findings in two animal models suggest that forced redirection of gene-regulatory elements may be used to alter gene expression to treat disease.

Lovo-cel gene therapy for sickle cell disease: Treatment process evolution and outcomes in the initial groups of the HGB-206 study.

lovo-cel (bb1111; LentiGlobin for sickle cell disease [SCD]) gene therapy (GT) comprises autologous transplantation of hematopoietic stem and progenitor cells transduced with the BB305 lentiviral vector encoding a modified ß-globin gene (ßA-T87Q ) to produce anti-sickling hemoglobin (HbAT87Q ). The efficacy and safety of lovo-cel for SCD are being evaluated in the ongoing phase 1/2 HGB-206 study (ClinicalTrials.gov: NCT02140554). The treatment process evolved over time, using learnings from outcomes in the initial patients to optimize lovo-cel's benefit-risk profile. Following modest expression of HbAT87Q in the initial patients (Group A, n = 7), alterations were made to the treatment process for patients subsequently enrolled in Group B (n = 2, patients B1 and B2), including improvements to cell collection and lovo-cel manufacturing. After 6 months, median Group A peripheral blood vector copy number (≥0.08 c/dg) and HbAT87Q levels (≥0.46 g/dL) were inadequate for substantial clinical effect but stable and sustained over 5.5 years; both markedly improved in Group B (patient B1: ≥0.53 c/dg and ≥2.69 g/dL; patient B2: ≥2.14 c/dg and ≥6.40 g/dL, respectively) and generated improved biologic and clinical efficacy in Group B, including higher total hemoglobin and decreased hemolysis. The safety of the lovo-cel for SCD treatment regimen largely reflected the known side effects of HSPC collection, busulfan conditioning regimen, and underlying SCD; acute myeloid leukemia was observed in two patients in Group A and deemed unlikely related to insertional oncogenesis. Changes made during development of the lovo-cel treatment process were associated with improved outcomes and provide lessons for future SCD GT studies.

Hematopoietic Stem Cell Gene-Addition/Editing Therapy in Sickle Cell Disease.

Autologous hematopoietic stem cell (HSC)-targeted gene therapy provides a one-time cure for various genetic diseases including sickle cell disease (SCD) and ß-thalassemia. SCD is caused by a point mutation (20A > T) in the ß-globin gene. Since SCD is the most common single-gene disorder, curing SCD is a primary goal in HSC gene therapy. ß-thalassemia results from either the absence or the reduction of ß-globin expression, and it can be cured using similar strategies. In HSC gene-addition therapy, patient CD34+ HSCs are genetically modified by adding a therapeutic ß-globin gene with lentiviral transduction, followed by autologous transplantation. Alternatively, novel gene-editing therapies allow for the correction of the mutated ß-globin gene, instead of addition. Furthermore, these diseases can be cured by γ-globin induction based on gene addition/editing in HSCs. In this review, we discuss HSC-targeted gene therapy in SCD with gene addition as well as gene editing.

Optimizing haematopoietic stem and progenitor cell apheresis collection from plerixafor-mobilized patients with sickle cell disease.

We adjusted haematopoietic stem and progenitor cell (HSPC) apheresis collection from patients with sickle cell disease (SCD) by targeting deep buffy coat collection using medium or low collection preference (CP), and by increasing anticoagulant-citrate-dextrose-solution A dosage. In 43 HSPC collections from plerixafor-mobilized adult patients with SCD, we increased the collection efficiency to 35.79% using medium CP and 82.23% using low CP. Deep buffy coat collection increased red blood cell contamination of the HSPC product, the product haematocrit was 4.7% with medium CP and 6.4% with low CP. These adjustments were well-tolerated and allowed efficient HSPC collection from SCD patients.

A macaque clonal hematopoiesis model demonstrates expansion of TET2-disrupted clones and utility for testing interventions.

Individuals with age-related clonal hematopoiesis (CH) are at greater risk for hematologic malignancies and cardiovascular diseases. However, predictive preclinical animal models to recapitulate the spectrum of human CH are lacking. Through error-corrected sequencing of 56 human CH/myeloid malignancy genes, we identified natural CH driver mutations in aged rhesus macaques matching genes somatically mutated in human CH, with DNMT3A mutations being the most frequent. A CH model in young adult macaques was generated via autologous transplantation of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated gene-edited hematopoietic stem and progenitor cells (HSPCs), targeting the top human CH genes with loss-of-function (LOF) mutations. Long-term follow-up revealed reproducible and significant expansion of multiple HSPC clones with heterozygous TET2 LOF mutations, compared with minimal expansion of clones bearing other mutations. Although the blood counts of these CH macaques were normal, their bone marrows were hypercellular and myeloid-predominant. TET2-disrupted myeloid colony-forming units isolated from these animals showed a distinct hyperinflammatory gene expression profile compared with wild type. In addition, mature macrophages purified from the CH macaques showed elevated NLRP3 inflammasome activity and increased interleukin-1ß (IL-1ß) and IL-6 production. The model was used to test the impact of IL-6 blockage by tocilizumab, documenting a slowing of TET2-mutated expansion, suggesting that interruption of the IL-6 axis may remove the selective advantage of mutant HSPCs. These findings provide a model for examining the pathophysiology of CH and give insights into potential therapeutic interventions.

Alemtuzumab clearance, lymphocyte count, and T-cell chimerism after hematopoietic stem cell transplant in sickle cell disease.

STUDY OBJECTIVE: Alemtuzumab is a monoclonal antibody that targets the cell surface antigen CD52 on lymphocytes. Although it is used for the treatment of hematologic malignancies, such as chronic lymphocytic leukemia, and incorporated into many hematopoietic stem cell transplant (HSCT) conditioning regimens, few studies have evaluated the pharmacology of alemtuzumab in adult patients with sickle cell disease (SCD). We therefore examined the pharmacokinetics (PK) and pharmacodynamics (PD) of alemtuzumab in adults with SCD who received a matched related donor HSCT to determine if the clearance of alemtuzumab affects transplant outcomes. DESIGN: PK and PD analysis of patient data from a single-center clinical trial. SETTING: Clinical research center. PATIENTS: Twenty-two adult patients with SCD who received one of two nonmyeloablative allogeneic HSCT regimens: alemtuzumab and total body irradiation (Alem-TBI) or pentostatin, cyclophosphamide, alemtuzumab, and total body irradiation (Pento-Cy-Alem-TBI). MEASUREMENTS AND MAIN RESULTS: Alemtuzumab serum concentrations, absolute lymphocyte counts, T-cell (CD3), and myeloid (CD14/15) chimerism were collected at distinct time points and analyzed. A semi-mechanistic PK population model was built to understand inter-individual differences in pharmacology. Alemtuzumab was detectable up to 28 days post-HSCT. The mean alemtuzumab level 7 days after transplant for patients on Alem-TBI was 818 ng/ml, significantly lower than the mean level of 1502 ng/ml for patients on Pento-Cy-Alem-TBI (p < 0.001), but this difference decreased as time progressed. The clearance of alemtuzumab was linear, and the half-life was longer in the Pento-Cy-Alem-TBI group (average half-life = 61.1 h) compared to the Alem-TBI group (average half-life = 44.1 h) (p < 0.001). The CD3 chimerism at 2 and 4 months after transplant positively correlated with alemtuzumab levels collected on day 14 after transplant (R2  = 0.40 and p = 0.004 at 2 months, R2  = 0.36 and p = 0.005 at 4 months), but this significance was lost by 6 months after HSCT. No correlation was seen between alemtuzumab levels and CD14/15 chimerism. CONCLUSION: Between 2 and 4 months after transplant, higher alemtuzumab levels measured 14 days after transplant correlated with patients having better engraftment, suggesting more lymphodepletion may be needed to reduce graft failure in these two non-myeloablative matched related donor HSCT regimens.

Disease severity impacts plerixafor-mobilized stem cell collection in patients with sickle cell disease.

Recent studies suggest that plerixafor mobilization and apheresis in patients with sickle cell disease (SCD) is safe and can allow collection of sufficient CD34+ hematopoietic stem cell (HSC) collection for clinical gene therapy applications. However, the quantities of plerixafor-mobilized CD34+ cells vary between different SCD patients for unknown reasons. Twenty-three participants with SCD underwent plerixafor mobilization followed by apheresis, processing, and HSC enrichment under a phase 1 safety and efficacy study conducted at 2 institutions. Linear regression or Spearman's correlation test was used to assess the relationships between various hematologic and clinical parameters with total CD34+ cells/kg collected. Median CD34+ cells/kg after 2 or fewer mobilization and apheresis cycles was 4.0 × 106 (range, 1.5-12.0). Similar to what is observed generally, CD34+ yield correlated negatively with age (P < .001) and positively with baseline (P = .003) and preapheresis blood CD34+ cells/µL (P < .001), and baseline white blood cell (P = .01) and platelet counts (P = .03). Uniquely for SCD, CD34+ cell yields correlated positively with the number of days hydroxyurea was held (for up to 5 weeks, P = .01) and negatively with markers of disease severity, including hospitalization frequency within the preceding year (P = .01) and the number of medications taken for chronic pain (P = .002). Unique SCD-specific technical challenges in apheresis were also associated with reduced CD34+ cell collection efficiency and purification. Here, we describe factors that impact plerixafor mobilization success in patients with SCD, confirming known factors as described in other populations in addition to reporting previously unknown disease specific factors in patients with SCD. This trial was registered at www.clinicaltrials.gov as #NCT03226691.

Preclinical evaluation for engraftment of CD34+ cells gene-edited at the sickle cell disease locus in xenograft mouse and non-human primate models.

Sickle cell disease (SCD) is caused by a 20A > T mutation in the ß-globin gene. Genome-editing technologies have the potential to correct the SCD mutation in hematopoietic stem cells (HSCs), producing adult hemoglobin while simultaneously eliminating sickle hemoglobin. Here, we developed high-efficiency viral vector-free non-footprint gene correction in SCD CD34+ cells with electroporation to deliver SCD mutation-targeting guide RNA, Cas9 endonuclease, and 100-mer single-strand donor DNA encoding intact ß-globin sequence, achieving therapeutic-level gene correction at DNA (∼30%) and protein (∼80%) levels. Gene-edited SCD CD34+ cells contributed corrected cells 6 months post-xenograft mouse transplant without off-target δ-globin editing. We then developed a rhesus ß-to-ßs-globin gene conversion strategy to model HSC-targeted genome editing for SCD and demonstrate the engraftment of gene-edited CD34+ cells 10-12 months post-transplant in rhesus macaques. In summary, gene-corrected CD34+ HSCs are engraftable in xenograft mice and non-human primates. These findings are helpful in designing HSC-targeted gene correction trials.

Cas9 protein delivery non-integrating lentiviral vectors for gene correction in sickle cell disease.

Gene editing with the CRISPR-Cas9 system could revolutionize hematopoietic stem cell (HSC)-targeted gene therapy for hereditary diseases, including sickle cell disease (SCD). Conventional delivery of editing tools by electroporation limits HSC fitness due to its toxicity; therefore, efficient and non-toxic delivery remains crucial. Integrating lentiviral vectors are established for therapeutic gene delivery to engraftable HSCs in gene therapy trials; however, their sustained expression and size limitation preclude their use for CRISPR-Cas9 delivery. Here, we developed a Cas9 protein delivery non-integrating lentiviral system encoding guide RNA and donor DNA, allowing for transient endonuclease function and inclusion of all editing tools in a single vector (all-in-one). We demonstrated efficient one-time correction of the SCD mutation in the endogenous ßs-globin gene up to 42% at the protein level (p < 0.01) with the Cas9 protein delivery non-integrating lentiviral all-in-one system without electroporation. Our findings improve prospects for efficient and safe genome editing.

Sustained fetal hemoglobin induction in vivo is achieved by BCL11A interference and coexpressed truncated erythropoietin receptor.

Hematopoietic stem cell gene therapy for hemoglobin disorders, including sickle cell disease, requires high-efficiency lentiviral gene transfer and robust therapeutic globin expression in erythroid cells. Erythropoietin is a key cytokine for erythroid proliferation and differentiation (erythropoiesis), and truncated human erythropoietin receptors (thEpoR) have been reported in familial polycythemia. We reasoned that coexpression of thEpoR could enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous nonhuman primate transplantation models. We generated thEpoR by deleting 40 amino acids from the carboxyl terminus, allowing for erythropoietin-dependent enhanced erythropoiesis of gene-modified cells. We then designed lentiviral vectors encoding both thEpoR and B cell lymphoma/leukemia 11A (BCL11A)-targeting microRNA-adapted short hairpin RNA (shmiR BCL11A) driven by an erythroid-specific promoter. thEpoR expression enhanced erythropoiesis among gene-modified cells in vitro. We then transplanted lentiviral vector gene-modified CD34+ cells with erythroid-specific expression of both thEpoR and shmiR BCL11A and compared to cells modified with shmiR BCL11A only. We found that thEpoR enhanced shmiR BCL11A-based fetal hemoglobin (HbF) induction in both xenograft mice and rhesus macaques, whereas HbF induction with shmiR BCL11A only was robust, yet transient. thEpoR/shmiR BCL11A coexpression allowed for sustained HbF induction at 20 to 25% in rhesus macaques for 4 to 8 months. In summary, we developed erythroid-specific thEpoR/shmiR BCL11A-expressing vectors, enhancing HbF induction in xenograft mice and rhesus macaques. The sustained HbF induction achieved by addition of thEpoR and shmiR BCL11A may represent a viable gene therapy strategy for hemoglobin disorders.

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.

Hematopoietic-Stem-Cell-Targeted Gene-Addition and Gene-Editing Strategies for ß-hemoglobinopathies.

Sickle cell disease (SCD) is caused by a well-defined point mutation in the ß-globin gene and therefore is an optimal target for hematopoietic stem cell (HSC) gene-addition/editing therapy. In HSC gene-addition therapy, a therapeutic ß-globin gene is integrated into patient HSCs via lentiviral transduction, resulting in long-term phenotypic correction. State-of-the-art gene-editing technology has made it possible to repair the ß-globin mutation in patient HSCs or target genetic loci associated with reactivation of endogenous γ-globin expression. With both approaches showing signs of therapeutic efficacy in patients, we discuss current genetic treatments, challenges, and technical advances in this field.