Sex-Dependent Transcriptional Changes in Response to Stress in Patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: A Pilot Project.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex, multi-symptom illness characterized by debilitating fatigue and post-exertional malaise (PEM). Numerous studies have reported sex differences at the epidemiological, cellular, and molecular levels between male and female ME/CFS patients. To gain further insight into these sex-dependent changes, we evaluated differential gene expression by RNA-sequencing (RNA-Seq) in 33 ME/CFS patients (20 female, 13 male) and 34 matched healthy controls (20 female and 14 male) before, during, and after an exercise challenge intended to provoke PEM. Our findings revealed that pathways related to immune-cell signaling (including IL-12) and natural killer cell cytotoxicity were activated as a result of exertion in the male ME/CFS cohort, while female ME/CFS patients did not show significant enough changes in gene expression to meet the criteria for the differential expression. Functional analysis during recovery from an exercise challenge showed that male ME/CFS patients had distinct changes in the regulation of specific cytokine signals (including IL-1ß). Meanwhile, female ME/CFS patients had significant alterations in gene networks related to cell stress, response to herpes viruses, and NF-κß signaling. The functional pathways and differentially expressed genes highlighted in this pilot project provide insight into the sex-specific pathophysiology of ME/CFS.

Stress-Induced Transcriptomic Changes in Females with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Reveal Disrupted Immune Signatures.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic, complex multi-organ illness characterized by unexplained debilitating fatigue and post-exertional malaise (PEM), which is defined as a worsening of symptoms following even minor physical or mental exertion. Our study aimed to evaluate transcriptomic changes in ME/CFS female patients undergoing an exercise challenge intended to precipitate PEM. Our time points (baseline before exercise challenge, the point of maximal exertion, and after an exercise challenge) allowed for the exploration of the transcriptomic response to exercise and recovery in female patients with ME/CFS, as compared to healthy controls (HCs). Under maximal exertion, ME/CFS patients did not show significant changes in gene expression, while HCs demonstrated altered functional gene networks related to signaling and integral functions of their immune cells. During the recovery period (commonly during onset of PEM), female ME/CFS patients showed dysregulated immune signaling pathways and dysfunctional cellular responses to stress. The unique functional pathways identified provide a foundation for future research efforts into the disease, as well as for potential targeted treatment options.

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.

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.

BCL11A enhancer-edited hematopoietic stem cells persist in rhesus monkeys without toxicity.

Gene editing of the erythroid-specific BCL11A enhancer in hematopoietic stem and progenitor cells (HSPCs) from patients with sickle cell disease (SCD) induces fetal hemoglobin (HbF) without detectable toxicity, as assessed by mouse xenotransplant. Here, we evaluated autologous engraftment and HbF induction potential of erythroid-specific BCL11A enhancer-edited HSPCs in 4 nonhuman primates. We used a single guide RNA (sgRNA) with identical human and rhesus target sequences to disrupt a GATA1 binding site at the BCL11A +58 erythroid enhancer. Cas9 protein and sgRNA ribonucleoprotein complex (RNP) was electroporated into rhesus HSPCs, followed by autologous infusion after myeloablation. We found that gene edits persisted in peripheral blood (PB) and bone marrow (BM) for up to 101 weeks similarly for BCL11A enhancer- or control locus-targeted (AAVS1-targeted) cells. Biallelic BCL11A enhancer editing resulted in robust γ-globin induction, with the highest levels observed during stress erythropoiesis. Indels were evenly distributed across PB and BM lineages. Off-target edits were not observed. Nonhomologous end-joining repair alleles were enriched in engrafting HSCs. In summary, we found that edited HSCs can persist for at least 101 weeks after transplant and biallelic-edited HSCs provide substantial HbF levels in PB red blood cells, together supporting further clinical translation of this approach.

ßT87Q-Globin Gene Therapy Reduces Sickle Hemoglobin Production, Allowing for Ex Vivo Anti-sickling Activity in Human Erythroid Cells.

Lentiviral addition of ßT87Q-globin, a modified ß-globin with an anti-sickling mutation, is currently being used in gene therapy trials for sickle cell disease (SCD) and ß-thalassemia patients. ßT87Q-globin interferes with sickle hemoglobin (HbS) polymerization. Here, we generated the SCD mutation in an immortalized human erythroid cell line (HUDEP-2) to investigate the anti-sickling activity of ßT87Q-globin. Sickle HUDEP-2 (sHUDEP-2) cells produced robust HbS after differentiation and sickled under deoxygenated conditions, comparable with SCD CD34+ progeny. Lentiviral transduction provided 9.5-26.8 pg/cell ßT87Q-globin (R2 = 0.83) in a vector copy number (VCN)-dependent manner, resulting in a significant reduction of sickling ratios (R2 = 0.92). Interestingly, ßT87Q-globin transduction markedly reduced endogenous ßS-globin (R2 = 0.84) to an undetectable level (0.4-16.8 pg/cell) in sHUDEP-2 cells, as well as endogenous ß-globin in human CD34+ cell-derived erythroid cells. RNA sequencing (RNA-seq) analysis with ßT87Q-transduced sHUDEP-2 and human CD34+-derived cells revealed activation of inflammation- and proliferation-related programs, suggesting minimal changes in background gene expression except for ßT87Q-globin expression and endogenous ß/ßS-globin suppression. In summary, using sHUDEP-2 and CD34+-derived cells, we demonstrated that lentiviral addition of ßT87Q-globin strongly reduced endogenous ß-/ßS-globin expression, resulting in an anti-sickling effect. Our findings should be helpful to understand the anti-sickling effects of therapeutic genes in SCD gene therapy.

Busulfan Combined with Immunosuppression Allows Efficient Engraftment of Gene-Modified Cells in a Rhesus Macaque Model.

Busulfan conditioning is utilized for hematopoietic stem cell (HSC) depletion in the context of HSC gene-therapy conditioning but may result in insufficient immunosuppression. In this study, we evaluated whether additional immunosuppression is required for efficient engraftment of gene-modified cells using a rhesus HSC lentiviral gene-therapy model. We transduced half of rhesus CD34+ cells with an enhanced green fluorescent protein (GFP)-encoding vector (immunogenic) and the other half with a γ-globin-encoding vector (no predicted immunogenicity). After autologous transplantation of both transduced cell populations following myeloablative busulfan conditioning (5.5 mg/kg/day for 4 days), we observed immunological rejection of GFP-transduced cells up to 3 months post-transplant and stable engraftment of γ-globin-transduced cells in two animals, demonstrating that ablative busulfan conditioning is sufficient for engraftment of gene-modified cells producing non-immunogenic proteins but insufficient to permit engraftment of immunogenic proteins. We then added immunosuppression with abatacept and sirolimus to busulfan conditioning and observed engraftment of both GFP- and γ-globin-transduced cells in two animals, demonstrating that additional immunosuppression allows for engraftment of gene-modified cells expressing immunogenic proteins. In conclusion, myeloablative busulfan conditioning should permit engraftment of gene-modified cells producing non-immunogenic proteins, while additional immunosuppression is required to prevent immunological rejection of a neoantigen.

Fetal hemoglobin and F-cell variance in mobilized CD34+ cell-transplanted rhesus monkeys.

Elevated fetal hemoglobin (HbF) is associated with reduced severity of sickle cell disease. Therefore, γ-globin protein levels and F-cell (HbF-positive red blood cell) percentages are used for estimation of clinical benefit. Here, we monitored transplantation-related changes in HbF and F-cell percentages for rhesus macaques (Macaca mulatta) following total body irradiation or busulfan conditioning prior to CD34+ cell transplantation. HbF protein expression peaked in the first 4-9 weeks posttransplant (0.99%-2.53%), and F-cells increased in the first 6-17 weeks posttransplant (8.7%-45.3%). HbF and F-cell ratios gradually decreased and stabilized to levels similar to those of control animals (1.96 ± 1.97% for F cells and 0.49 ± 0.19% γ-globin expression) 4-7 months post-transplant. These findings confirm and expand on previous reports of transient induction in HbF and F-cell percentages in rhesus macaques following CD34+ cell transplantation, an observation that must be taken into consideration when evaluating therapeutic strategies that aim to specifically elevate HbF expression, which are currently in clinical development.

High-Efficiency Lentiviral Transduction of Human CD34+ Cells in High-Density Culture with Poloxamer and Prostaglandin E2.

Hematopoietic stem cell (HSC) gene therapy is curative for various hereditary diseases; however, high-efficiency transduction in HSCs remains crucial to improve the prospects for hemoglobinopathies. We previously optimized lentiviral transduction in human CD34+ cells with serum-free medium containing minimal cytokines, allowing efficient transduction (∼50%) and robust xenograft engraftment. In this study, we further improved lentiviral transduction in human CD34+ cells. High-density culture conditions (4e6/mL) resulted in ∼5-fold more efficient transduction in CD34+ cells (p < 0.01) compared with standard cell density (1e5/mL). After co-culturing vector-exposed CD34+ cells with non-transduced CD34+ cells, high-density culture conditions enhanced lentiviral gene marking in the non-transduced population (p < 0.01) compared with low-density conditions, suggesting that increasing cell-to-cell contact allows more efficient transduction. Two adjuvants, poloxamer 407 (100 µg/mL) and prostaglandin E2 (10 µM), were added to high-density CD34+ cells, resulting in ∼4-fold more efficient transduction (p < 0.01) without significant toxicity compared with no adjuvant control. In summary, we developed a highly efficient lentiviral transduction method in high-density CD34+ cell culture with poloxamer 407 and prostaglandin E2, allowing overall ∼10-fold improvement in transduction efficiency and consistently achieving more than 90% transduction and an average vector copy number of ∼10. Our optimized transduction method should improve gene therapy approaches using lentiviral vectors targeting HSCs.

Low-Dose Busulfan Reduces Human CD34+ Cell Doses Required for Engraftment in c-kit Mutant Immunodeficient Mice.

Humanized animal models are central to efforts aimed at improving hematopoietic stem cell (HSC) transplantation with or without genetic modification. Human cell engraftment is feasible in immunodeficient mice; however, high HSC doses and conditioning limit broad use of xenograft models. We assessed human CD45+ chimerism after transplanting varying doses of human CD34+ HSCs (2 × 105 to 2 × 106 cells/mouse) with or without busulfan (BU) pretransplant conditioning in c-kit mutant mice that do not require conditioning (non-obese diabetic [NOD]/B6/severe combined immunodeficiency [SCID]/ interleukin-2 receptor gamma chain null (IL-2rγ-/-) KitW41/W41 [NBSGW]). We then tested a range of BU (5-37.5 mg/kg) using 2 × 105 human CD34+ cells. Glycophorin-A erythrocyte chimerism was assessed after murine macrophage depletion using clodronate liposomes. We demonstrated successful long-term engraftment of human CD34+ cells at all cell doses in this model, and equivalent engraftment using 10-fold less CD34+ cells with the addition of BU conditioning. Low-dose BU (10 mg/kg) was sufficient to allow human engraftment using 2 × 105 CD34+ cells, whereas higher doses (≥37.5 mg/kg) were toxic. NBSGW mice support human erythropoiesis in the bone marrow; however, murine macrophage depletion provided only minimal and transient increases in peripheral blood human erythrocytes. Our xenograft model is therefore useful in HSC gene therapy and genome-editing studies, especially for modeling in disorders, such as sickle cell disease, where access to HSCs is limited.

The Role of Bmp2 in the Maturation and Maintenance of the Murine Knee Joint.

Bone morphogenetic proteins (BMPs) are key regulators of skeletal development, growth, and repair. Although BMP signaling is required for synovial joint formation and is also involved in preserving joint function after birth, the role of specific BMP ligands in adult joint homeostasis remains unclear. The purpose of this study was to define the role of Bmp2 in the morphogenesis and maintenance of the knee joint. To do this, we first created Bmp2-LacZ and Gdf5-LacZ knock-in mice and compared their expression patterns in the developing and postnatal murine knee joint. We then generated a knockout mouse model using the Gdf5-cre transgene to specifically delete Bmp2 within synovial joint-forming cells. Joint formation, maturation, and homeostasis were analyzed using histology, immunohistochemistry, qRT-PCR, and atomic force microscopy (AFM)-based nanoindentation to assess the cellular, molecular, and biomechanical changes in meniscus and articular cartilage. Bmp2 is expressed in the articular cartilage and meniscus of the embryonic and adult mouse knee in a pattern distinct from Gdf5. The knee joints of the Bmp2 knockout mice form normally but fail to mature properly. In the absence of Bmp2, the extracellular matrix and shape of the meniscus are altered, resulting in functional deficits in the meniscus and articular cartilage that lead to a progressive osteoarthritis (OA) like knee pathology as the animals age. These findings demonstrate that BMP activity provided by Bmp2 is required for the maturation and maintenance of the murine knee joint and reveal a unique role for Bmp2 that is distinct from Gdf5 in knee joint biology. © 2018 American Society for Bone and Mineral Research.

Identification and characterization of adult mouse meniscus stem/progenitor cells.

Meniscal damage is a common problem that accelerates the onset of knee osteoarthritis. Stem cell-based tissue engineering treatment approaches have shown promise in preserving meniscal tissue and restoring meniscal function. The purpose of our study was to identify meniscus-derived stem/progenitor cells (MSPCs) from mouse, a model system that allows for in vivo analysis of the mechanisms underlying meniscal injury and healing. MSPCs were isolated from murine menisci grown in explant culture and characterized for stem cell properties. Flow cytometry was used to detect the presence of surface antigens related to stem cells, and qRT-PCR was used to examine the gene expression profile of MSPCs. Major proteins associated with MSPCs were localized in the adult mouse knee using immunohistochemistry. Our data show that MSPCs have universal stem cell-like properties including clonogenicity and multi-potentiality. MSPCs expressed the mesenchymal stem cell markers CD44, Sca-1, CD90, and CD73 and when cultured had elevated levels of biglycan and collagen type I, important extracellular matrix components of adult meniscus. MSPC also expressed significant levels of Lox and Igf-1, genes associated with the embryonic meniscus. Localization studies showed staining for these same proteins in the superficial and outer zones of the adult mouse meniscus, regions thought to harbor endogenous repair cells. MSPCs represent a novel resident stem cell population in the murine meniscus. Analysis of MSPCs in mice will allow for a greater understanding of the cell biology of the meniscus, essential information for enhancing therapeutic strategies for treating knee joint injury and disease.