Progenitor Cell Transplantation Research Articles

Autologous bone marrow-derived mesenchymal stem cells and endothelial progenitor cells transplantation showed potential benefits for type 2 diabetes mellitus Filipino patients: a case series

Autologous bone marrow-derived mesenchymal stem cells and endothelial progenitor cells transplantation showed potential benefits for type 2 diabetes mellitus Filipino patients: a case series

Neural Stem Cell-based Regenerative Therapy: A New Approach to Diabetes Treatment.

Diabetes mellitus (DM) is the most common metabolic disorder that occurs due to the loss, or impaired function of insulin-secreting pancreatic beta cells, which are of two types - type 1 (T1D) and type 2 (T2D). To cure DM, the replacement of the destroyed pancreatic beta cells of islet of Langerhans is the most widely practiced treatment. For this, isolating neuronal stem cells and cultivating them as a source of renewable beta cells is a significant breakthrough in medicine. The functions, growth, and gene expression of insulin-producing pancreatic beta cells and neurons are very similar in many ways. A diabetic patient's neural stem cells (obtained from the hippocampus and olfactory bulb) can be used as a replacement source of beta cells for regenerative therapy to treat diabetes. The same protocol used to create functional neurons from progenitor cells can be used to create beta cells. Recent research suggests that replacing lost pancreatic beta cells with autologous transplantation of insulin-producing neural progenitor cells may be a perfect therapeutic strategy for diabetes, allowing for a safe and normal restoration of function and a reduction in potential risks and a long-term cure.

Clickable Granular Hydrogel Scaffolds for Delivery of Neural Progenitor Cells to Sites of Spinal Cord Injury.

Spinal cord injury (SCI) is a serious condition with limited treatment options. Neural progenitor cell (NPC) transplantation is a promising treatment option, and the identification of novel biomaterial scaffolds that support NPC engraftment and therapeutic activity is a top research priority. The objective of this study is to evaluate in situ assembled poly (ethylene glycol) (PEG)-based granular hydrogels for NPC delivery in a murine model of SCI. Microgel precursors are synthesized by using thiol-norbornene click chemistry to react four-armed PEG-amide-norbornene with enzymatically degradable and cell adhesive peptides. Unreacted norbornene groups are utilized for in situ assembly into scaffolds using a PEG-di-tetrazine linker. The granular hydrogel scaffolds exhibit good biocompatibility and do not adversely affect the inflammatory response after SCI. Moreover, when used to deliver NPCs, the granular hydrogel scaffolds supported NPC engraftment, do not adversely affect the immune response to the NPC grafts, and successfully support graft differentiation toward neuronal or astrocytic lineages as well as axonal extension into the host tissue. Collectively, these data establish PEG-based granular hydrogel scaffolds as a suitable biomaterial platform for NPC delivery and justify further testing, particularly in the context of more severe SCI.

Evaluation of benefits and risks of immunosuppressive drugs in biomaterial-based neural progenitor cell transplantation for spinal cord injury repair

Spinal cord injury (SCI) can disrupt neural connections and impair locomotion and sensation function. Currently, biomaterials containing neural progenitor cells (NPCs) are widely used to replace damaged tissue or provide support for neural regeneration following SCI. Immunosuppressive drugs (ISDs) are commonly utilized to ensure the survival of transplanted NPC in SCI. However, the potential benefits and risks of using ISDs in NPC transplantation with biomaterials for SCI repair has not been deeply studied. Herein, an orderly aligned collagen sponge scaffold (ACSS) combined with human embryonic spinal cord neural progenitor cells (hscNPCs) was transplanted into SCI rats, with or without ISDs. Results showed that ISDs suppressed cell-mediated immune and inflammatory responses, maintained early survival and differentiation capacity of transplanted cells, and promoted early functional recovery of SCI rats. However, ISDs did not influence endogenous nerve regeneration, angiogenesis, glial scarring or long-term functional recovery after ACSS and hscNPC transplantation. Additionally, ISDs can induce toxicity in multiple organ and impact the survival of animals after SCI. Thus, careful evaluation of the benefits and risks is necessary to maximize therapeutic efficiency when administrating ISD in combination with scaffold-loaded NPCs for SCI repair.

Lung Progenitor and Stem Cell Transplantation as a Potential Regenerative Therapy for Lung Diseases.

Chronic lung diseases are debilitating illnesses ranking among the top causes of death globally. Currently, clinically available therapeutic options capable of curing chronic lung diseases are limited to lung transplantation, which is hindered by donor organ shortage. This highlights the urgent need for alternative strategies to repair damaged lung tissues. Stem cell transplantation has emerged as a promising avenue for regenerative treatment of the lung, which involves delivery of healthy lung epithelial progenitor cells that subsequently engraft in the injured tissue and further differentiate to reconstitute the functional respiratory epithelium. These transplanted progenitor cells possess the remarkable ability to self-renew, thereby offering the potential for sustained long-term treatment effects. Notably, the transplantation of basal cells, the airway stem cells, holds the promise for rehabilitating airway injuries resulting from environmental factors or genetic conditions such as cystic fibrosis. Similarly, for diseases affecting the alveoli, alveolar type II cells have garnered interest as a viable alveolar stem cell source for restoring the lung parenchyma from genetic or environmentally induced dysfunctions. Expanding upon these advancements, the use of induced pluripotent stem cells to derive lung progenitor cells for transplantation offers advantages such as scalability and patient specificity. In this review, we comprehensively explore the progress made in lung stem cell transplantation, providing insights into the current state of the field and its future prospects.

Donor KIR2DL1 Allelic Polymorphism Influences Posthematopoietic Progenitor Cell Transplantation Outcomes in the T Cell Depleted and Reduced Intensity Conditioning Setting

The majority of established KIR clinical assessment algorithms used for donor selection for hematopoietic progenitor cell transplantation (HPCT) evaluate gene content (presence/absence) of the KIR gene complex. In comparison, relatively little is known about the impact of KIR allelic polymorphism. By analyzing donors of T cell depleted (TcD) reduced intensity conditioning (RIC) HPCT, this study investigated the influence on post-transplant outcome of 2 polymorphic residues of the inhibitory KIR2DL1. The aim of this study was to expand upon existing research into the influence of KIR2DL1 allelic polymorphism upon post-transplant outcome. The effects of allele groups upon transplant outcomes were investigated within a patient cohort using a defined treatment protocol of RIC with TcD. Using phylogenetic data, KIR2DL1 allelic polymorphism was categorized into groups on the basis of variation within codons 114 and 245 (positive or negative for the following groups: KIR2DL1*002/001g, KIR2DL1*003, KIR2DL1*004g) and the identification of null alleles. The influence of these KIR2DL1 allele groups in hematopoietic progenitor cell transplantation (HPCT) donors was assessed in the post-transplant data of 86 acute myelogenous leukemia patients receiving RIC TcD HPCT at a single center. KIR2DL1 allele groups in the donor significantly impacted upon 5-year post-transplant outcomes in RIC TcD HPCT. Donor KIR2DL1*003 presented the greatest influence upon post-transplant outcomes, with KIR2DL1*003 positive donors severely reducing 5-year post-transplant overall survival (OS) compared to those receiving a transplant from a KIR2DL1*003 negative donor (KIR2DL1*003 pos versus neg: 27.0% versus 60.0%, P = .008, pc = 0.024) and disease-free survival (DFS) (KIR2DL1*003 pos versus neg: 23.5% versus 60.0%, P = .004, pc = 0.012), and increasing 5-year relapse incidence (KIR2DL1*003 pos versus neg: 63.9% versus 27.2%, P = .009, pc = 0.027). KIR2DL1*003 homozygous and KIR2DL1*003 heterozygous grafts did not present significantly different post-transplant outcomes. Donors possessing the KIR2DL1*002/001 allele group were found to significantly improve post-transplant outcomes, with donors positive for the KIR2DL1*004 allele group presenting a trend towards improvement. KIR2DL1*002/001 allele group (KIR2DL1*002/001g) positive donors improved 5-year OS (KIR2DL1*002/001g pos versus neg: 56.4% versus 27.2%, P = .009, pc = 0.024) and DFS (KIR2DL1*002/001g pos versus neg: 53.8% versus 25.5%, P = .018, pc = 0.036). KIR2DL1*004 allele group (KIR2DL1*004g) positive donors trended towards improving 5-year OS (KIR2DL1*004g pos versus neg: 53.3% versus 35.5%, P = .097, pc = 0.097) and DFS (KIR2DL1*004g pos versus neg: 50.0% versus 33.9%, P = .121, pc = 0.121), and reducing relapse incidence (KIR2DL1*004g pos versus neg: 33.1% versus 54.0%, P = .079, pc = 0.152). The presented findings suggest donor selection algorithms for TcD RIC HPCT should consider avoiding KIR2DL1*003 positive donors, where possible, and contributes to the mounting evidence that KIR assessment in donor selection algorithms should reflect the conditioning regime protocol used.

Autologous transplantation of P63+ lung progenitor cells for chronic obstructive pulmonary disease therapy.

Adult lung resident stem/progenitor cells, including P63+ progenitor cells, have demonstrated the capacity for regeneration of lung epithelium in preclinical models. Here, we report a clinical trial of intrapulmonary P63+ progenitor cell transplantation in 28 participants with stage II to IV chronic obstructive pulmonary disease (COPD). Autologous P63+ progenitor cells were isolated from the airway basal layer of participants in the intervention group via bronchoscopic brushing, cultured for 3 to 5 weeks, and then transplanted back into the lungs via bronchoscopy at 0.7 × 106 to 5.2 × 106 cells per kilogram of body weight. Twenty patients were evaluable at the end of the study (intervention group, n = 17; control group, n = 3). No grade 3 to 5 adverse events (AEs) or serious AEs occurred. Although bronchoscopy-associated AEs were recorded in participants in the intervention group, other AEs were not substantial different between groups. Twenty-four weeks after transplantation, participants in the intervention group displayed improvement in gas transfer capacity [diffusing capacity of the lung for carbon monoxide (DLCO) change from baseline: +18.2%], whereas the control group experienced a decrease (DLCO change from baseline: -17.4%; P = 0.008). Furthermore, participants in the intervention group showed >30-meter increase in walking distance within 6 minutes. Transcriptomic analysis of progenitor cells isolated from responding and nonresponding individuals in the intervention group showed that higher expression of P63 was associated with treatment efficacy. In conclusion, transplantation of cultured P63+ lung progenitor cells was safe and might represent a potential therapeutic strategy for COPD.

Label-Free and High-Throughput Removal of Residual Undifferentiated Cells From iPSC-Derived Spinal Cord Progenitor Cells.

The transplantation of spinal cord progenitor cells (SCPCs) derived from human-induced pluripotent stem cells (iPSCs) has beneficial effects in treating spinal cord injury (SCI). However, the presence of residual undifferentiated iPSCs among their differentiated progeny poses a high risk as these cells can develop teratomas or other types of tumors post-transplantation. Despite the need to remove these residual undifferentiated iPSCs, no specific surface markers can identify them for subsequent removal. By profiling the size of SCPCs after a 10-day differentiation process, we found that the large-sized group contains significantly more cells expressing pluripotent markers. In this study, we used a sized-based, label-free separation using an inertial microfluidic-based device to remove tumor-risk cells. The device can reduce the number of undifferentiated cells from an SCPC population with high throughput (ie, >3 million cells/minute) without affecting cell viability and functions. The sorted cells were verified with immunofluorescence staining, flow cytometry analysis, and colony culture assay. We demonstrated the capabilities of our technology to reduce the percentage of OCT4-positive cells. Our technology has great potential for the "downstream processing" of cell manufacturing workflow, ensuring better quality and safety of transplanted cells.

Exonic knockout and knockin gene editing in hematopoietic stem and progenitor cells rescues RAG1 immunodeficiency.

Recombination activating genes (RAGs) are tightly regulated during lymphoid differentiation, and their mutations cause a spectrum of severe immunological disorders. Hematopoietic stem and progenitor cell (HSPC) transplantation is the treatment of choice but is limited by donor availability and toxicity. To overcome these issues, we developed gene editing strategies targeting a corrective sequence into the human RAG1 gene by homology-directed repair (HDR) and validated them by tailored two-dimensional, three-dimensional, and in vivo xenotransplant platforms to assess rescue of expression and function. Whereas integration into intron 1 of RAG1 achieved suboptimal correction, in-frame insertion into exon 2 drove physiologic human RAG1 expression and activity, allowing disruption of the dominant-negative effects of unrepaired hypomorphic alleles. Enhanced HDR-mediated gene editing enabled the correction of human RAG1 in HSPCs from patients with hypomorphic RAG1 mutations to overcome T and B cell differentiation blocks. Gene correction efficiency exceeded the minimal proportion of functional HSPCs required to rescue immunodeficiency in Rag1-/- mice, supporting the clinical translation of HSPC gene editing for the treatment of RAG1 deficiency.

Long Term Remission of Capillary Leak Syndrome Associated with Monoclonal Gammopathy with Progression to Multiple Myeloma After Autologous Stem Cell Transplantation: a Case Report and Review of the Literature.

Clarkson's disease is a very rare entity characterised by acute episodes of systemic oedema and severe hypotension associated with paraproteinaemia. Its classical treatment relies on methylxanthine combined with terbutaline. Although this prophylactic therapy reduces the mortality rate, relapses are frequent. Eighty percent of patients with Clarkson's disease present with monoclonal gammopathy of unknown significance (MGUS). The risk of progression to multiple myeloma is 1% per year. Here, we present a 49-year-old woman who suffered multiple such episodes requiring treatment in the intensive care unit. Treatment with terbutaline and theophylline was ineffective. She was diagnosed with multiple myeloma (MM) 8 years after the first of these acute episodes. Antimyeloma treatment with bortezomib and dexamethasone was started, followed by autologous haemopoietic transplantation, with no further acute episodes since then. Our case is, to our knowledge, unique because eradication of MM was followed by complete disappearance of acute episodes of capillary leakage. Our case report is also the first to support the use of bortezomib and dexamethasone in this setting. Furthermore, autologous peripheral blood progenitor cell transplantation consolidated the MM stringent complete remission achieving a very long progression-free survival (> 11 years) of both MM and Clarkson's disease.

α7nAChR Activation Combined with Endothelial Progenitor Cell Transplantation Attenuates Lung Injury in Diabetic Rats with Sepsis through the NF-κB Pathway.

Chronic diabetes mellitus compromises the vascular system, which causes organ injury, including in the lung. Due to the strong compensatory ability of the lung, patients always exhibit subclinical symptoms. Once sepsis occurs, the degree of lung injury is more severe under hyperglycemic conditions. The α7 nicotinic acetylcholine receptor (α7nAChR) plays an important role in regulating inflammation and metabolism and can improve endothelial progenitor cell (EPC) functions. In the present study, lung injury caused by sepsis was compared between diabetic rats and normal rats. We also examined whether α7nAChR activation combined with EPC transplantation could ameliorate lung injury in diabetic sepsis rats. A type 2 diabetic model was induced in rats via a high-fat diet and streptozotocin. Then, a rat model of septic lung injury was established by intraperitoneal injection combined with endotracheal instillation of LPS. The oxygenation indices, wet-to-dry ratios, and histopathological scores of the lungs were tested after PNU282987 treatment and EPC transplantation. IL-6, IL-8, TNF-α, and IL-10 levels were measured. Caspase-3, Bax, Bcl-2, and phosphorylated NF-κB (p-NF-κB) levels were determined by blotting. Sepsis causes obvious lung injury, which is exacerbated by diabetic conditions. α7nAChR activation and endothelial progenitor cell transplantation reduced lung injury in diabetic sepsis rats, alleviating inflammation and decreasing apoptosis. This treatment was more effective when PNU282987 and endothelial progenitor cells were administered together. p-NF-κB levels decreased following treatment with PNU282987 and EPCs. In conclusion, α7nAChR activation combined with EPC transplantation can alleviate lung injury in diabetic sepsis rats through the NF-κB signaling pathway.

Small extracellular vesicles derived from human induced pluripotent stem cell-differentiated neural progenitor cells mitigate retinal ganglion cell degeneration in a mouse model of optic nerve injury.

JOURNAL/nrgr/04.03/01300535-202502000-00034/figure1/v/2024-05-28T214302Z/r/image-tiff Several studies have found that transplantation of neural progenitor cells (NPCs) promotes the survival of injured neurons. However, a poor integration rate and high risk of tumorigenicity after cell transplantation limits their clinical application. Small extracellular vesicles (sEVs) contain bioactive molecules for neuronal protection and regeneration. Previous studies have shown that stem/progenitor cell-derived sEVs can promote neuronal survival and recovery of neurological function in neurodegenerative eye diseases and other eye diseases. In this study, we intravitreally transplanted sEVs derived from human induced pluripotent stem cells (hiPSCs) and hiPSCs-differentiated NPCs (hiPSC-NPC) in a mouse model of optic nerve crush. Our results show that these intravitreally injected sEVs were ingested by retinal cells, especially those localized in the ganglion cell layer. Treatment with hiPSC-NPC-derived sEVs mitigated optic nerve crush-induced retinal ganglion cell degeneration, and regulated the retinal microenvironment by inhibiting excessive activation of microglia. Component analysis further revealed that hiPSC-NPC derived sEVs transported neuroprotective and anti-inflammatory miRNA cargos to target cells, which had protective effects on RGCs after optic nerve injury. These findings suggest that sEVs derived from hiPSC-NPC are a promising cell-free therapeutic strategy for optic neuropathy.

WBP1L regulates hematopoietic stem cell function and T cell development.

WW domain binding protein 1-like (WBP1L), also known as outcome predictor of acute leukemia 1 (OPAL1), is a transmembrane adaptor protein, expression of which was shown to correlate with ETV6-RUNX1 translocation and favorable prognosis in childhood leukemia. It has a broad expression pattern in hematopoietic and non-hematopoietic cells. Our previous work described WBP1L as a regulator of CXCR4 signaling and hematopoiesis. Here, we show that hematopoiesis in the mice with Wbp1l germline deletion is dysregulated, already at the level of hematopoietic stem cells and early progenitors. We further demonstrate that thymi of WBP1L-deficient mice are significantly enlarged and contain increased numbers of thymocytes of all subsets. This can potentially be explained by increased generation of multipotent progenitors 4 (MPP4) in the bone marrow, from which the thymus-seeding progenitors are derived. We also observed increases in multiple cell types in the blood. In addition, we show that WBP1L regulates hematopoietic stem cell functionality and leukocyte progenitor proliferation and gene expression during hematopoietic stem and progenitor cell transplantation, which contribute to more efficient engraftment of WBP1L-deficient cells. WBP1L thus emerges as a regulator of hematopoietic stem and progenitor cell function, which controls leukocyte numbers at the steady state and after bone marrow transplantation.

Identification and validation of novel engineered AAV capsid variants targeting human glia.

Direct neural conversion of endogenous non-neuronal cells, such as resident glia, into therapeutic neurons has emerged as a promising strategy for brain repair, aiming to restore lost or damaged neurons. Proof-of-concept has been obtained from animal studies, yet these models do not efficiently recapitulate the complexity of the human brain, and further refinement is necessary before clinical translation becomes viable. One important aspect is the need to achieve efficient and precise targeting of human glial cells using non-integrating viral vectors that exhibit a high degree of cell type specificity. While various naturally occurring or engineered adeno-associated virus (AAV) serotypes have been utilized to transduce glia, efficient targeting of human glial cell types remains an unsolved challenge. In this study, we employ AAV capsid library engineering to find AAV capsids that selectively target human glia in vitro and in vivo. We have identified two families of AAV capsids that induce efficient targeting of human glia both in glial spheroids and after glial progenitor cell transplantation into the rat forebrain. Furthermore, we show the robustness of this targeting by transferring the capsid peptide from the parent AAV2 serotype onto the AAV9 serotype, which facilitates future scalability for the larger human brain.

Ongoing production of tissue-resident macrophages from hematopoietic stem cells in healthy adult macaques

AbstractMacrophages orchestrate tissue immunity from the initiation and resolution of antimicrobial immune responses to the repair of damaged tissue. Murine studies demonstrate that tissue-resident macrophages are a heterogenous mixture of yolk sac–derived cells that populate the tissue before birth, and bone marrow–derived replacements recruited in adult tissues at steady-state and in increased numbers in response to tissue damage or infection. How this translates to species that are constantly under immunologic challenge, such as humans, is unknown. To understand the ontogeny and longevity of tissue-resident macrophages in nonhuman primates (NHPs), we use a model of autologous hematopoietic stem progenitor cell (HSPC) transplantation with HSPCs genetically modified to be marked with clonal barcodes, allowing for subsequent analysis of clonal ontogeny. We study the contribution of HSPCs to tissue macrophages, their clonotypic profiles relative to leukocyte subsets in the peripheral blood, and their transcriptomic and epigenetic landscapes. We find that HSPCs contribute to tissue-resident macrophage populations in all anatomic sites studied. Macrophage clonotypic profiles are dynamic and overlap significantly with the clonal hierarchy of contemporaneous peripheral blood monocytes. Epigenetic and transcriptomic landscapes of HSPC-derived macrophages are similar to tissue macrophages isolated from NHPs that did not undergo transplantation. We also use in vivo bromodeoxyuridine infusions to monitor tissue macrophage turnover in NHPs that did not undergo transplantation and find evidence for macrophage turnover at steady state. These data demonstrate that the life span of most tissue-resident macrophages is limited and can be replenished continuously from HSPCs.

CD33-Deleted Hematopoietic Cells (Trem-Cel) Are Protected from CD33xCD3 Bispecific Antibody Treatment and Produce Significantly Reduced Levels of Inflammatory Cytokines in Preclinical Studies

INTRODUCTION: Acute myeloid leukemia (AML) is the most common form of adult acute leukemia. Currently, allogeneic hematopoietic stem cell transplant (HCT) is the standard of care for high-risk patients yet ~40% patients will relapse, emphasizing the need for new therapeutic approaches. Effective targeted therapy in AML has been hampered by a lack of cancer specific targets. One described AML target is CD33, a glycoprotein expressed predominantly on normal myeloid cells and the majority of AML cells. However, CD33-directed therapies have been challenged by cytopenias due to on-target, off-tumor toxicity and, in the case of bispecific antibodies, cytokine release syndrome (CRS). Tremtelectogene empogeditemcel (trem-cel) is a gene-edited hematopoietic stem and progenitor cell (HSPC) transplant product currently in clinical trial (NCT04849910) whereby CD33, shown to be dispensable for normal hematopoiesis and function, is deleted to allow for post-HCT treatment with CD33-directed immunotherapies with reduced myelotoxicity as the reconstituted hematopoietic compartment lacks CD33. JNJ-67571244, a bispecific antibody that binds to both CD3 and CD33, is capable of inducing T cell recruitment for CD33-directedtumor cytotoxicity. This study examines whether CD33 neg hematopoietic cells are protected from JNJ-67571244 and if CD33 neg human HSPC (hHSPC) xenotransplanted mice have reduced levels of inflammatory cytokines associated with CRS when treated with JNJ-67571244. METHODS: In vitro proof-of-concept (POC) studies were carried out using CD3 + T cells co-cultured with JNJ-67571244 and AML target cell lines. Further studies were conducted with CD34 + hHSPCs that were either CRISPR/Cas9 gene edited to delete CD33 or treated with a non-targeting gRNA control (gCtrl) followed by differentiation towards the monocytic lineage. These in vitro differentiated monocytes (MIVD) were assayed for editing frequency, CD33protein expression and myeloid phenotype by flow cytometry prior to co-culture with donor matched T cells and JNJ-67571244 ranging from 0.5 pM to 500 nM. Cytotoxicity was analyzed using flow cytometry at 48 and 72 hr and cytokine concentrations in the supernatants were measured using Luminex. For in vivo POC studies, CD33 or gCtrl-edited CD34 + hHSPCs were xenotransplanted into NSG-SGM3 mice followed by treatment with either control bispecific (CD3xNull) or JNJ-67571244 at 0.5 mg/kg. Plasma cytokine concentration were measured via Luminex and hematopoietic tissues were analyzed by flow cytometry to measure protection from JNJ-67571244 cytotoxic killing. RESULTS: Annexin V and Live/Dead staining showed that JNJ-67571244 was able to induce AML target cell killing by human T cells. Importantly CD33 neg MIVD cells co-cultured with T cells and JNJ-67571244 were significantly more viable at all doses compared to gCtrl cells. The EC 50 of gCtrl MIVD was 0.0208 nM whereas the CD33 neg MIVD EC 50 was at least 20,000-fold higher (>430.8 nM). T cell activation, measured by CD25 and CD69 positivity, and release of cytokines that are associated with CRS such as IFN-γ, TNF-α, IL-2, IL-6 and IL-10, were all significantly decreased in the CD33 neg MIVD cells treated with JNJ-67571244 compared to the gCtrl group at all doses. Analysis of blood and bone marrow from xenotransplant studies revealed that JNJ-67571244 was effective at killing CD33 + cells as well as CD14 + monocytes in the gCtrl group treated with JNJ-67571244. Conversely, CD14 + monocytes and CD15 + neutrophils derived from CD33-edited hHSPCs, were not significantly reduced in cell number when treated with JNJ-67571244 suggesting that they were protected from JNJ-67571244 specific killing. IFN-γ, TNF-α, IL-2, IL-6 and IL-10, were all significantly decreased in mice transplanted with CD33-edited hHSPCs compared to gCtrl group in response to JNJ-67571244. CONCLUSIONS: Taken together, these studies demonstrate that CD33 deleted hematopoietic compartment is protected from the CD33 directed immuno-therapy JNJ-67571244 both in in vitro cytotoxicity assays and preclinical xenotransplantation studies, with decreased concentrations of inflammatory cytokines associated with CRS. These findings enable the development of a next-generation AML treatment strategy by pairing trem-cel transplant with a subsequent CD33-directed bispecific compound to potentially improve safety and efficacy while minimizing myelotoxicity.

Novel Small Molecule VLA-4 Inhibitors Optimized for Extended Hematopoietic and Progenitor Cell Mobilization

Background: Hematopoietic stem and progenitor cell (HSPC) transplant is an essential treatment for a variety of blood disorders and malignancies, and more recently, for gene editing therapies such as sickle cell anemia. A key step in this procedure is the effective mobilization of a large quantity of primitive donor HSPCs to ensure a durable and long lasting engraftment capable of developing into all blood cell lineages. The most commonly used regimen for donor mobilization is a 5-day course of Granulocyte Colony Stimulating Factor (G-CSF). However, G-CSF does not effectively mobilize a sufficient number or quality of stem cells in all donors, can have unwanted side effects, and is contraindicated in sickle cell disease. Therefore, it is desirable to identify an alternative treatment that is safe, rapid and cost effective. The cell surface integrin receptor VLA-4 (a4b1) and the chemokine receptor CXCR4 are essential for HSPC homing and retention in the bone marrow. Inhibition of these receptors rapidly mobilizes HSPCs within an hour, as opposed to the multi-day process using G-CSF. Small molecule inhibitors of VLA-4 have been reported previously, but most have poor pharmacokinetic properties and/or weak inhibition of VLA4. These inhibitors rapidly mobilize HSPCs into the peripheral blood of mice after a single s.c. injection, but their effect is short lived, yielding an inadequate number of HSPCs necessary for stem cell transplantation or for gene therapy. Therefore, VLA4 inhibitors with improved pharmacokinetic properties imparting extended and greater HSPC mobilization after a single dose is desirable. Methods: We designed and synthesized multiple novel VLA-4 inhibitor molecules and tested their potency using soluble VCAM-1 binding assays. Inhibitors determined to be most potent with improved aqueous solubility were then tested in vivo in DBA mice for extended HSPC mobilization beyond 4 hours after a single SC injection, alone and in combination with the CXCR4 inhibitors. Results: We developed two main core molecules that each incorporate key VLA4 receptor binding motifs critical for potent inhibition of VLA4 binding to VCAM-1. Our first generation of inhibitors were quite potent and rapidly mobilized HSPCs, but their effect was short lived and yielded insufficient numbers of HSPCs for gene editing and engraftment studies. Most had poor solubility and very short plasma half-lives after s.c. injection in mice. We designed later generation analogues that strategically exploited a region of each optimized core, allowing for covalent addition of polyethylene glycol (PEG) chains of increasing length without decreasing potency. We determined that a minimum PEG length of 24 PEG units was required for extended HSPC mobilization in mice. WU-106, one of our VLA-4 inhibitors containing 24 PEG units, exhibited excellent aqueous solubility, longer plasma half-life after s.c. dosing, and mobilized 2-fold more murine HSPCs for a longer time than previously published best-in-class VLA-4 inhibitors or our first generation VLA4 inhibitor leads. Importantly, a synergistic effect on HSPC mobilization was achieved when WU-106 was co-administered with CXCR4 inhibitors (Plerixafor or BL-8040). Further optimization by the addition of longer PEG chains, even up to 40KD, retained sub nM potency and extended mobilization beyond 24 hours after a single injection. Our 20kD PEG analogues attached to our two different core molecules (WU-125 and WU-133) allowed for dosing of the drug in the evening, with maximum mobilization still present the next morning, thereby enabling HSPC collection to initiate soon (2-4 hours) after CXCR4 inhibitor dosing in the morning. The quantity of HSPCs obtained using this staggered dosing regimen was greater than simultaneous administration of the VLA-4 and CXCR4 inhibitors ( Fig. 1). Furthermore, our novel, long-acting PEG analogues demonstrated superior HSPC mobilization compared to an alpha 4-targeted antibody (anti-CD49d) in mice ( Fig.2). Conclusion: We have developed novel, potent PEGylated small molecule VLA-4 inhibitors that have excellent aqueous solubility and pharmacokinetics. The kinetics and magnitude of HSPC mobilization by our PEGylated inhibitors are superior to other previously described VLA-4 inhibitors and an antibody to alpha-4(anti-CD49d).

Preclinical Assessment for Gene Addition Strategy in Familial Hemophagocytic Lymphohistiocytosis Related to Munc 13-4 Deficiency

Introduction: Familial Hemophagocytic Lymphohistiocytosis (FHL) comprises a group of rare genetic disorders with defect cytotoxicity in T and NK cells predisposing to severe hyperinflammation with the clinical hallmark of the Hemophagocytic LymphoHistiocytosis (HLH) syndrome. The uncontrolled proliferation of activated T-CD8 lymphocytes and macrophages infiltrates lymphoid tissues, bone marrow, and multiple organs including the brain. Thus, if untreated full-scale FHL is usually fatal. FHL3 is due to mutations in the UNC13D gene coding for Munc 13-4 protein, one of the components of the perforin-dependent cytotoxicity apparatus. This condition accounts for 30% to 35 % of all genetic cases of HLH. Hematopoietic stem and progenitor cell (HSPC) transplantation, which is the only curative treatment for FHL3 to date, remains difficult even when a compatible donor is available because of the patients' critical inflammatory background. In this context, gene therapy could be a promising therapeutic option, especially for those patients without HLA-compatible donor. Method: The feasibility and efficacy of gene addition strategy for FHL3 have been previously demonstrated in mouse models. We then translated this approach to human cells using a third-generation self-inactivated lentivirus vector pseudo-typed with a conventional vesicular stomatitis virus-G (VSVG) expressing a codon-optimized UNC13D gene under the transcriptional control of EF1 alpha (LV-EF1a-UNC13D). HSPC or CD3+ T-cells were transduced with a GMP-like-grade LV-EF1a-UNC13D lentiviral vector. We studied the restoration of cytotoxic function. Results: Our GMP transduction protocol led to efficient transcription of optimized mRNA codon and expression of Munc 13-4 protein as evidenced by RT qPCR and western blot, respectively. Interestingly, we show restored cytotoxicity activity after CD34+ differentiation toward CD3+ T-cells and NK cells. The vector copy number is compatible with a clinical approach. In vivo experiments are ongoing to validate the absence of genotoxicity in this setting. Discussion conclusion: Our results support the feasibility and efficacy of UNCD13D gene transfer into hematopoietic stem and progenitor cells or T cells from Munc 13-4 deficient patients. Alternatively, gene therapy of HSPCs could be combined with transduced autologous T-cells infusion. The first attempts regarding this option are encouraging. UNC13D gene addition strategy could dramatically improve the care of FHL3 patients lacking an HLA-genoidentical donor.

Nod1-dependent NF-kB activation initiates hematopoietic stem cell specification in response to small Rho GTPases

Uncovering the mechanisms regulating hematopoietic specification not only would overcome current limitations related to hematopoietic stem and progenitor cell (HSPC) transplantation, but also advance cellular immunotherapies. However, generating functional human induced pluripotent stem cell (hiPSC)-derived HSPCs and their derivatives has been elusive, necessitating a better understanding of the developmental mechanisms that trigger HSPC specification. Here, we reveal that early activation of the Nod1-Ripk2-NF-kB inflammatory pathway in endothelial cells (ECs) primes them to switch fate towards definitive hemogenic endothelium, a pre-requisite to specify HSPCs. Our genetic and chemical embryonic models show that HSPCs fail to specify in the absence of Nod1 and its downstream kinase Ripk2 due to a failure on hemogenic endothelial (HE) programming, and that small Rho GTPases coordinate the activation of this pathway. Manipulation of NOD1 in a human system of definitive hematopoietic differentiation indicates functional conservation. This work establishes the RAC1-NOD1-RIPK2-NF-kB axis as a critical intrinsic inductor that primes ECs prior to HE fate switch and HSPC specification. Manipulation of this pathway could help derive a competent HE amenable to specify functional patient specific HSPCs and their derivatives for the treatment of blood disorders.

Comparative study of the efficacy of intra-arterial and intravenous transplantation of human induced pluripotent stem cells-derived neural progenitor cells in experimental stroke.

Cell therapy using neural progenitor cells (NPCs) is a promising approach for ischemic stroke treatment according to the results of multiple preclinical studies in animal stroke models. In the vast majority of conducted animal studies, the therapeutic efficacy of NPCs was estimated after intracerebral transplantation, while the information of the effectiveness of systemic administration is limited. Nowadays, several clinical trials aimed to estimate the safety and efficacy of NPCs transplantation in stroke patients were also conducted. In these studies, NPCs were transplanted intracerebrally in the subacute/chronic phase of stroke. The results of clinical trials confirmed the safety of the approach, however, the degree of functional improvement (the primary efficacy endpoint) was not sufficient in the majority of the studies. Therefore, more studies are needed in order to investigate the optimal transplantation parameters, especially the timing of cell transplantation after the stroke onset. This study aimed to evaluate the therapeutic effects of intra-arterial (IA) and intravenous (IV) administration of NPCs derived from induced pluripotent stem cells (iNPCs) in the acute phase of experimental stroke in rats. Induced pluripotent stem cells were chosen as the source of NPCs as this technology is perspective, has no ethical concerns and provides the access to personalized medicine. Human iNPCs were transplanted IA or IV into male Wistar rats 24 h after the middle cerebral artery occlusion stroke modeling. Therapeutic efficacy was monitored for 14 days and evaluated in comparison with the cell transplantation-free control group. Additionally, cell distribution in the brain was assessed. The obtained results show that both routes of systemic transplantation (IV and IA) significantly reduced the mortality and improved the neurological deficit of experimental animals compared to the control group. At the same time, according to the MRI data, only IA administration led to faster and prominent reduction of the stroke volume. After IA administration, iNPCs transiently trapped in the brain and were not detected on day 7 after the transplantation. In case of IV injection, transplanted cells were not visualized in the brain. The obtained data demonstrated that the systemic transplantation of human iNPCs in the acute phase of ischemic stroke can be a promising therapeutic strategy.