Allogeneic Hematopoietic Stem Cell Research Articles

Lessons Learned From Ocular Graft versus Host Disease: An Ocular Surface Inflammatory Disease of Known Time of Onset.

The ocular surface inflammatory disorders (OSIDs) comprise a group of conditions characterized by persistent inflammation of the ocular surface and adnexal tissues. Systemic autoimmune diseases and hypersensitivity reactions cause them, and, if left untreated, can result in severe inflammatory dry eye, corneal damage, and vision loss. Ocular graft-versus-host disease (oGVHD) forms part of the ocular surface inflammatory disease umbrella. It is a condition occurring after allogeneic hematopoietic stem cell or bone marrow transplantation, usually in chronic graft-versus-host disease. oGVHD can virtually affect any ocular adnexal tissue, especially the meibomian glands, and cause persistent inflammation, tissue fibrosis, and subsequent chronic, severe dry eye disease. Among the OSIDs, oGVHD has the particularity that it has a "time zero," meaning we know when the disease started. As such, preclinical models have leveraged this to investigate the molecular mechanisms involved in the damage oGVHD causes to the ocular surface. In oGVHD, establishing a "time zero" allows for predicting the clinical course and establishing adequate treatment. This is also possible because the inflammatory infiltration occurs in ocular surface tissues, which are readily accessible. Using oGVHD, we might be able to understand the immune response mechanisms in other OSIDs better (i.e., Sjögren syndrome, Stevens-Johnson syndrome, among others). This review presents an up-to-date overview of the pathogenesis, clinical presentation, and treatment of oGVHD. In addition, we will discuss the value of the "time zero" concept in the study of oGVHD.

Secondary solid malignancies and precancerous lesions after allogeneic hematopoietic stem cell transplantation using non-total body irradiation-based conditioning in acute myeloid leukemia

IntroductionLong-term survivors have an increased risk of developing secondary solid malignancies (SSMs) after allogeneic-hematopoietic stem cell transplantation (allo-HSCT) with graft-versus-host disease (GVHD) potentially modulating these risks.MethodsThis retrospective study analyzed the cumulative incidences of SSMs after chemotherapy-based conditioning for allo-HSCT patients with acute myeloid leukemia (n = 266) transplanted at the University Hospital Regensburg between 1999 and 2016.ResultsThe median follow-up was 11.4 years (Interquartile range, 9.0–14.9). The 100-day cumulative incidence of grade II-IV acute GVHD (aGVHD) was 44.4% [95% CI (38.3, 50.2)], while the 5-year cumulative incidence of chronic GVHD (cGVHD, requiring systemic immunosuppression) was 36.9% [95% CI (31.1, 42.6)]. The cumulative incidences of secondary squamous cell carcinomas (SCCs) at 10 and 15 years were 4.2% [95% CI (2.2, 7.2)] and 8.1% [95% CI (4.6, 12.8)], while the cumulative incidences of non-SCCs at 10 and 15 years were 5.4% [95% CI (3.1, 8.7)] and 6.9% [95% CI (4.0, 10.8)]. Antithymocyte globulin (ATG) was associated with reduced incidences of SCCs but not of non-SCCs. Patients with grade II-IV aGVHD had increased rates of SCCs after adjusting with patient age and ATG, while patients with cGVHD showed only a trend for increased rates of SCCs.ConclusionThe data indicate that aGVHD and cGVHD affect the rates of secondary SCCs. While the use of ATG is associated with lower incidences of SCCs via reduction of GVHD, there was no association of ATG with non-SCCs.

Human herpes virus type 6 (Orthoherpesviridae: <i>Roseolovirus</i>): features of epidemiology and diagnosis

Human herpes virus 6A and human herpes virus 6B (HHV-6A and HHV-6B) are ubiquitous viruses. The spectrum of clinical manifestations of HHV-6A/B infections is quite wide. The current understanding of the natural history and laboratory diagnosis of HHV-6A and HHV-6B, including their chromosome-integrated form, serves the basis for development of the tools for HHV-6 epidemiological monitoring. This article addresses the epidemiology and diagnosis of infections caused by these viruses, including ones in patients after transplantation of solid organs and allogeneic hematopoietic stem cells.

Anti-CD45 PBD-based antibody-drug conjugates are effective targeted conditioning agents for gene therapy and stem cell transplant

Stem cell gene therapy and hematopoietic stem cell transplantation (SCT) and stem cell gene therapy require conditioning to ablate the recipient's hematopoietic stem cells (HSC) and create a niche for gene-corrected/donor HSCs. Conventional conditioning agents are non-specific, leading to off-target toxicities, resulting in significant morbidity and mortality. We developed tissue-specific anti-human CD45 antibody-drug conjugates (ADCs), using rat IgG2b anti-human CD45 antibody clones YTH24.5 and YTH54.12, conjugated to cytotoxic pyrrolobenzodiazepine (PBD) dimer payloads with cleavable (SG3249) or non-cleavable (SG3376) linkers. In vitro, these ADCs internalized to lysosomes for drug release, resulting in potent and specific killing of human CD45+ cells. In humanized NSG mice, the ADCs completely ablated human HSCs without toxicity to non-hematopoietic tissues, enabling successful engraftment of gene-modified autologous and allogeneic human HSCs. The ADCs also delayed leukemia onset and improved survival in CD45+ tumor models. These data provide proof-of-concept that conditioning with anti-human CD45-PBD ADCs allows engraftment of donor/gene-corrected HSC, with minimal toxicity to non-hematopoietic tissues. Our anti-CD45-PBDs or similar agents could potentially shift the paradigm in transplantation medicine that intensive chemo/radiotherapy is required for HSC engraftment after gene therapy and allogeneic SCT. Targeted conditioning both improve the safety and minimize late-effects of these procedures which would greatly increase their applicability.

Myelofibrosis: Current unmet needs, emerging treatments, and future perspectives.

The current standard-of-care for treatment of myelofibrosis (MF) comprises inhibitors of the Janus kinase (JAK)/signal transducers and activators (STAT) pathway; however, despite their ability to alleviate symptoms, they do not appear to modify underlying disease and have not demonstrated substantial survival benefit. Allogeneic-hematopoietic stem cell transplantation remains the only curative option for patients with MF but is limited to a subset of high-risk and fit patients. Early disease modification could positively affect disease trajectory for lower risk patients with MF as well as those with conditions that can precede MF, such as polycythemia vera and essential thrombocythemia. Here, the authors discuss critical unmet needs in the MF treatment paradigm, including: the need for safe, impactful therapies for lower risk patients, thus allowing intervention when success is most likely; better development of first-line therapies (likely highly novel or combination strategies) for intermediate-risk/higher risk patients; and approved drugs to manage cytopenia. Finally, a consensus definition of disease modification is needed that informs trial design, allowing the development of clinical end points that enable understanding of therapies and responses and that facilitate the development of therapies that work according to this definition. Through close collaboration between clinicians, patients, and the pharmaceutical industry, better efforts to define benefit and identify patients most likely to benefit from a particular combination or treatment strategy should enable the development of more effective and safe treatments to extend and improve quality of life for patients with MF.

Perspectives for the Use of Umbilical Cord Blood in Transplantation and Beyond: Initiatives for an Advanced and Sustainable Public Banking Program in Greece.

The umbilical cord blood (UCB) donated in public UCB banks is a source of hematopoietic stem cells (HSC) alternative to bone marrow for allogeneic HSC transplantation (HSCT). However, the high rejection rate of the donated units due to the strict acceptance criteria and the wide application of the haploidentical HSCT have resulted in significant limitation of the use of UCB and difficulties in the economic sustainability of the public UCB banks. There is an ongoing effort within the UCB community to optimize the use of UCB in the field of HSCT and a parallel interest in exploring the use of UCB for applications beyond HSCT i.e., in the fields of cell therapy, regenerative medicine and specialized transfusion medicine. In this report, we describe the mode of operation of the three public UCB banks in Greece as an example of an orchestrated effort to develop a viable UCB banking system by (a) prioritizing the enrichment of the national inventory by high-quality UCB units from populations with rare human leukocyte antigens (HLA), and (b) deploying novel sustainable applications of UCB beyond HSCT, through national and international collaborations. The Greek paradigm of the public UCB network may become an example for countries, particularly with high HLA heterogeneity, with public UCB banks facing sustainability difficulties and adds value to the international efforts aiming to sustainably expand the public UCB banking system.

The impact of cryoprotectant exposure time on post-thaw viability of autologous and allogeneic hematopoietic stem cells and leukocyte subpopulations.

Although the use of cryoprotectant dimethyl sulfoxide (DMSO) is the gold standard in cryopreservation of hematopoietic stem cells, it is well known that it has a negative effect on cell viability. The aim of this prospective study was to examine how the length of post-thaw exposure to DMSO affects the cell viability and stability of peripheral blood stem cell (PBSC) samples. Additionally, the effects of donor type and pre-cryopreservation storage time on post-thaw viability during the stability study were evaluated. In 30 autologous and 30 allogeneic PBSC samples viable CD34+, CD14+, CD19+, CD16+/56+, and CD3+ cells were determined immediately after thawing, and one-and three-hours post-thaw. Analysis of the absolute count of viable cells in thawed samples showed a significant difference between all measurement points for CD34+ (p < 0.001), CD14+ (p < 0.001), and CD19+ cells (p < 0.001). No significant differences were observed for post-thaw stability of allogeneic samples analysed between products stored before cryopreservation ≥ 24 hours (N = 20), and those stored < 24 hours (N = 10), except for viable CD3+/CD4+ cells after three hours post-thaw (p = 0.028). In conclusion, DMSO had different effects on leukocyte subpopulations in cryopre-served PBSC samples. The type of donors and the length of storage before cryopreservation did not affect the post-thaw stability of cryopreserved PBSC samples.

Hematopoietic Stem Cells from Mice and Individuals with Sickle Cell Disease Display Premature Senescence and Loss of Function That Is Targetable By Senolytic Therapy

Sickle cell disease (SCD) is an inherited anemia caused by mutations in the β-globin gene, resulting in aberrant hemoglobin polymerization driving hemolysis, vasooclusion, and systemic inflammation. Allogeneic hematopoietic stem cell (HSC) transplantation is potentially curative, and novel autologous therapies involving gene-editing of mobilized HSCs are being developed. However, many SCD patients are unable to undergo autologous gene-editing due to failure to mobilize sufficient numbers of HSCs. Further, the damaging effects of SCD on the HSC pool remain uncharacterized. Therefore, we interrogated young and aged cohorts of humanized SCD mice and non-SCD controls for HSC frequency and function. Immunophenotypic HSC frequency was mildly elevated (1.3-fold, p=0.00316) in 2-month-old SCD mice, relative to controls (n=5-6/group). In contrast, HSC numbers were halved in 6 and 12-month-old SCD mice (n=8-10/group, p=0.00358 and p=0.0148, respectively). Competitive transplantation of whole bone marrow (WBM) from 2 and 6-month-old SCD mice displayed a profound loss of peripheral blood repopulation, relative to age-matched control mice. Quantitative limiting dilution transplantation of WBM from 2 and 6-month-old SCD mice revealed a 4 and 6-fold loss of functional HSCs, relative to control mice. Thus, despite elevated numbers of phenotypic HSCs in young SCD mice, the HSC pool is depleted of functional repopulating cells during SCD. To understand the loss of functional HSCs, we performed bulk RNA-sequencing of purified HSCs from 6-month-old SCD mice. We observed downregulation of 37 genes encoding ribosomal and histone proteins, suggesting impaired histone/ribosome biogenesis, a phenotype associated with senescence. Consistently, pathway enrichment analysis also revealed perturbed p53 activity and a high degree of association with published molecular signatures of senescence. To explore this further, HSCs isolated from 6-month-old SCD mice or age-matched controls were interrogated for senescence associated β-galactosidase (SA-β-gal) activity. On average 19.5% of SCD HSCs were SA-β-gal+ versus only 7.06% of HSCs isolated from controls (n=10-11/group, p=0.004). HSCs isolated from SCD mice also displayed elevated ROS, DNA damage, and expression of molecular markers of senescence (e.g. p21, BCLXL). Importantly, 23.14 ± 5.61% of Lin-CD34+CD38- bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) isolated from pediatric individuals with SCD were SA-β-gal+. In contrast, only 4.97 ± 2.97% of HSPCs were SA-β-gal+ in age-matched, non-SCD control individuals (n=8 control and n=5 SCD individuals, p=&amp;lt;0.0001). Further, 47.46 ± 17.49% of Lin-CD34+CD38-CD90+CD45RA- BM cells, which are highly enriched for human HSCs, were SA-β-gal+ in young individuals with SCD, relative to only 4.41 ± 2.37% in the BM of age-matched control individuals (n=4 control and n=5 SCD individuals, p=0.0019). We also detected elevated expression of p16, p21, and BCL2 in BM HSPCs isolated from pediatric individuals with SCD, relative to controls (n=6 control and n=9 SCD individuals). In total, these data reveal that SCD pathophysiology aberrantly invokes senescence programs and damages HSC function in mice and humans. Clearance of senescent HSCs from mouse BM has previously been shown to restore aging-related HSC dysfunction. We therefore tested if clearance of senescent HSCs from the BM could restore function to the SCD HSC pool. 2-month-old SCD and control mice were treated with two weekly cycles of 75 mg/kg/day of the senolytic, ABT-263, via oral gavage. 14 days after treatment, WBM was isolated and tested for HSC function and frequency. Senolytic therapy significantly increased HSC numbers in SCD mice BM, relative to vehicle-treated controls (n=5/group). Importantly, the in vivo hematopoietic repopulating activity of WBM from ABT-263-treated SCD mice was comparable to non-SCD controls, revealing a correction of our previously observed severe transplantation defect (n=12/group). In sum, these data reveal premature onset of senescence as a previously unappreciated phenotype of the HSC pool during SCD. This work provides the first proof-of-principle that treatment of individuals with SCD using senolytics may restore function to the HSC pool, and ongoing preclinical work aims to test if senolytic therapy enhances the yield and function of SCD HSCs mobilized for autologous therapy.

Prime Editing Efficiently and Precisely Corrects Causative Mutation in Chronic Granulomatous Disease, Restoring Myeloid Function: Toward Development of a Prime Edited Autologous Hematopoietic Stem Cell Therapy

Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency characterized by a lack of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in phagocytic myeloid cells such as neutrophils. NADPH oxidase produces reactive oxygen species (ROS) that directly kill bacteria and fungi to control infection. CGD patients lack this ability to control infection, which results in chronic and severe infections, inflammation, autoimmunity, poor quality of life, and reduced lifespan. Current standard care for CGD is antimicrobial prophylaxis and aggressive management of infection. For patients with a suitable human leukocyte antigen (HLA) matched donor, an allogeneic hematopoietic stem cell (HSC) transplant (allo-HSCT) offers a curative approach. However, many patients do not have access to HLA matched donors and allo-HSCT also carries a risk of GvHD and graft rejection. P47phox-deficient CGD, the most common autosomal recessive CGD, is caused by mutations in the NCF1 gene, which encodes the p47phox protein, a subunit of the NADPH oxidase complex. Approximately 80% of p47phox-deficient CGD patients carry the same homozygous mutation, a 2 nucleotide GT deletion (delGT) in exon 2 of NCF1 which is correctable using Prime Editing. Transplantation of Prime Edited patient autologous CD34 + cells, in which the delGT mutation has been corrected with Prime Editing, may provide a curative treatment for p47phox-deficient CGD patients. To evaluate whether correction of the delGT NCF1 mutation in CGD patient CD34 + cells restores p47phox protein expression and NADPH oxidase activity in myeloid progeny, Prime Editors were designed to correct this mutation precisely and efficiently. The Prime Editors were delivered to CD34 + cells from two p47phox-deficient CGD patients homozygous for delGT NCF1. In both studies, ≧75% of Prime Edited patient CD34 + cells carried at least 1 corrected allele as determined by ddPCR and DNA sequencing analyses. Myeloid progeny differentiated from Prime Edited patient CD34 + cells had restored p47phox protein expression and NADPH oxidase activity to ~80% of normal healthy donors as determined by multiple ROS detection assays. To evaluate reproducibility, Prime Editors were delivered to CD34 + cells from 6 different healthy donors. On average, greater than 90% of CD34 + cells were Prime Edited across 6 donors in independent experiments. To confirm that Prime Edited CD34 + cells retain stem cell multipotency, engraftment, and hematopoietic reconstitution properties, Prime Edited and mock treated CD34 + cells from three different donors were transplanted into immunodeficient mice. Sixteen weeks after CD34 + cell transplantation, &amp;gt;87% of long-term hematopoietic stem cells (LT-HSC) that repopulated the bone marrow had at least 1 allele corrected. There were no significant differences between ‘electroporation only’ mock treated and Prime Editor electroporated human CD34 + cells with respect to long-term CD34 + cell engraftment, human CD45 + blood cell chimerism, hematopoietic multilineage blood cell reconstitution and biodistribution, or LT-HSC cell potency. Across these studies, &amp;gt;95% human CD45 + cell chimerism was achieved with Prime Edited CD34 + cells. Despite high on-target correction of the delGT mutation in NCF1, off-target editing, unintended editing, and chromosomal rearrangements (larger deletions or translocation) events were not detected using a robust suite of genome-wide assays. In summary, Prime Editing corrects a mutation that causes the most common autosomal recessive from of CGD; correction of this delGT mutation at NCF1 restores NADPH oxidase activity and function in myeloid progeny of Prime Edited CGD CD34 + cells; and Prime Edited long-term HSCs retain multipotency and functionality in vivo. These results support the development of a Prime Edited CGD patient autologous CD34 + cell therapy as a potential curative approach for p47phox-deficient CGD patients.

Cryopreservation Impacts Allogenic Stem Cell Engraftment: Relation between Graft Quality Control and Post-Transplant Clinical Outcome

Introduction: Freezing hematopoietic stem cells (HSC) has been a standard procedure in cellular therapy, either for allogenic, autologous, or cord blood transplantation. While cryopreservation is generally reported in the literature as a safe procedure with little impact on graft quality, some reports suggest significant effects of cryopreserved HSC as compared to fresh cells in the outcome of Hematopoietic Stem Cell Transplantation (HSCT). Recently, the COVID-19 pandemic enforced cryopreservation of HSC to secure allograft transplantation from related and unrelated donors. Modifying our standard practices was a unique opportunity to analyze the impact of this additional procedure on cell quality and HSC engraftment. Method: This study compared the HSCT engraftment from fresh versus cryopreserved allogeneic HSC performed in Necker Hospital (n = 236) between January 2020 to December 2022. We retrospectively collected clinical data on patients from our unicentric PROMISE database and extracted the graft characteristics (cell phenotypes, viability, and clonogenicity) from the quality assurance database. Delayed platelets or neutrophils recoveries, early graft loss, and the need for CD34+ selected stem cell boost were used to define post-transplant graft dysfunctions. Results: 107 pediatric patients with a median age of 3.9 years old and 129 Adult patients underwent primary HSCT in our center during the COVID-19 pandemic. Indications were hematological malignancies (n= 97: Acute leukemia, myelodysplastic syndrome, lymphoma, plasma cell disorders), inborn error diseases (n= 131: primary immune deficiency, hemoglobin disorder, histiocytic disorder), and bone marrow failure (n= 8). Sixty six (28.0%) graft of hematopoietic stem cells were cryopreserved from Bone Marrow (BM) (n = 18, 14.8%) and Peripheral Blood Stem Cells (PBSC) (n = 48, 42.1%). Cryopreservation significantly affects the viability (98.2% fresh vs. 84.4% cryopreserved HSC, p&amp;lt;0.0001) and the clonogenicity (18.7% fresh vs. 8.75% cryopreserved HSC, p&amp;lt;0.0001), whatever the source of stem cells. While cell viability and clonogenicity complied with the international standard before graft infusion, graft dysfunctions were a significant concern for cryopreserved allogeneic transplant (n= 22; 33.3%). Graft dysfunctions in cryopreserved HSC groups occurred with both bone marrow and PBSC. Significantly, dysfunctions increased to 38.5% for cryopreserved BM despite a higher median dose of CD34+/kg infused 15.5 (range, 10.2-20.7). No quality control parameter was predictive of engraftment dysfunction. Discussion conclusion: Cryopreservation of HSC was critical during the SARS-CoV-2 pandemic; however, in our experience, the procedure could impact graft function. Considering the development and worldwide distribution of cell therapy drug products, accurately evaluating the pretransplant graft quality is an issue in predicting the engraftment of HSC.

Expression of a large coding sequence: Gene therapy vectors for Ataxia Telangiectasia

Ataxia telangiectasia is a monogenetic disorder caused by mutations in the ATM gene. Its encoded protein kinase ATM plays a fundamental role in DNA repair of double strand breaks (DSBs). Impaired function of this kinase leads to a multisystemic disorder including immunodeficiency, progressive cerebellar degeneration, radiation sensitivity, dilated blood vessels, premature aging and a predisposition to cancer. Since allogenic hematopoietic stem cell (HSC) transplantation improved disease outcome, gene therapy based on autologous HSCs is an alternative promising concept. However, due to the large cDNA of ATM (9.2 kb), efficient packaging of retroviral particles and sufficient transduction of HSCs remains challenging.We generated lentiviral, gammaretroviral and foamy viral vectors with a GFP.F2A.Atm fusion or a GFP transgene and systematically compared transduction efficiencies. Vector titers dropped with increasing transgene size, but despite their described limited packaging capacity, we were able to produce lentiviral and gammaretroviral particles. The reduction in titers could not be explained by impaired packaging of the viral genomes, but the main differences occurred after transduction. Finally, after transduction of Atm-deficient (ATM-KO) murine fibroblasts with the lentiviral vector expressing Atm, we could show the expression of ATM protein which phosphorylated its downstream substrates (pKap1 and p-p53).

Early reversal of the lymphocyte-to-monocyte ratio after allogeneic-hematopoietic stem cell transplantation is associated with reduced relapse and improved prognosis.

The early recovery of lymphocyte and monocyte cells is associated with a favorable prognosis after allogeneic stem cell transplantation (allo-HSCT); however, it is not clear whether the balance of lymphocyte and monocyte recovery affects the post-transplant prognosis. We examined whether the time-point at which the number of lymphocytes exceeded the number of monocytes, which we termed lymphocyte-to-monocyte ratio reversal (LMRR), affected the prognosis after allo-HSCT. We retrospectively evaluated 235 patients who underwent their first allo-HSCT at our institution. The median number of days from HSCT to LMRR was 46 (range, 0-214), and the patients were divided into two groups according to the occurrence of LMRR by day 45 (LMRR45). In a multivariate analysis, early LMRR contributed favorably to overall survival (hazard ratio [HR] .519; 95% confidence interval [CI] .332-.812; p=.004) with fewer post-transplant relapses (HR .462; 95% CI, .274-.777; p=.004). Differences in the timing of LMRR did not affect non-relapse mortality (HR 1.477; 95% CI .779-2.80; p=.23) or the incidence of grade II-IV acute GVHD (LMRR45(+): 25.0% vs. LMRR45(-) 35.2%. p=.111). In subgroup analyses, LMRR45(+) was found to be a favorable factor for survival with less relapse, regardless of the disease risk, stem cell source, or the recovery of either lymphocyte or monocyte counts. An early LMRR may be a novel factor that is associated with reduced relapse and improved survival after allo-HSCT.

Use of subgroup-specific hematopoietic stem cell collection efficiencies to improve truncation calculations for large-volume leukapheresis procedures.

A critical component of optimizing peripheral blood (PB) hematopoietic stem cell (HSC) collections is accurately determining the processed blood volume required to collect the targeted number of HSCs. Fundamental to most truncation equations employed to determine this volume is the procedure's estimated collection efficiency (CE), which is typically applied uniformly across all HSC collections. Few studies have explored the utility of using different CEs in subpopulations of donors that have substantially different CEs than the institutional average. Initial procedures from 343 autologous and 179 allogeneic HSC collections performed from 2018 to 2021 were retrospectively analyzed. Predictive equations were developed to determine theoretical truncation rates in various donor subgroups. Quantitative variables (pre-procedure cell counts) and qualitative variables (relatedness to recipient, gender, method of venous access, and mobilization strategy) were found to significantly impact CE. However, much of the variability in CE between donors could not be explained by the variables assessed. Analyses of procedures with high pre-collection PB cell counts identified lower CE values for these donors' truncation equations which still allow truncation but minimize risk of collecting less CD34+ cells than requested. Individualized CE does not substantially improve truncation volume calculations over use of a fixed CE and adds complexity to these calculations. The optimal fixed CE varies between autologous and allogeneic donors, and donors with high pre-collection PB cell counts in either of these groups. This model will be clinically validated and continuously refined through analysis of future HSC collections.

Physioxia improves the selectivity of hematopoietic stem cell expansion cultures.

Hematopoietic stem cells (HSCs) are a rare hematopoietic cell type that can entirely reconstitute the blood and immune systems following transplantation. Allogeneic HSC transplantation (HSCT) is used clinically as a curative therapy for a range of hematolymphoid diseases, but remains a high-risk therapy due to potential side effects including poor graft function and graft-vs-host disease (GvHD). Ex vivo HSC expansion has been suggested as an approach to improve hematopoietic reconstitution from low-cell dose grafts. Here, we demonstrate that we can improve the selectivity of polyvinyl alcohol (PVA)-based mouse HSC cultures through the use of physioxic culture conditions. Single-cell transcriptomic analysis confirmed inhibition of lineage-committed progenitor cells in physioxic cultures. Long-term physioxic expansion also afforded culture-based ex vivo HSC selection from whole bone marrow, spleen, and embryonic tissues. Furthermore, we provide evidence that HSC-selective ex vivo cultures deplete GvHD-causing T cells and that this approach can be combined with genotoxic-free antibody-based conditioning HSCT approaches. Our results offer a simple approach to improve PVA-based HSC cultures and the underlying molecular phenotype, as well as highlight the potential translational implications of selective HSC expansion systems for allogeneic HSCT.

Sterility Testing for Hematopoietic Stem Cells.

Over the last two decades, rapid technological advances have led to the wide adoption of cell and gene therapy products for the treatment of a variety of disease states. In this study, we reviewed the literature between 2003 and 2021 to provide a summary of overarching trends associated with microbial contamination in hematopoietic stem cells (HSCs) derived from peripheral blood, bone marrow, and cord blood. We provide a brief background on the regulatory context for human cells, tissues, and cellular and tissue-based products (HCT/Ps) as regulated by the US Food and Drug Administration (FDA), sterility testing expectations for autologous (Section 361) and allogeneic (Section 351) HSC products, and discuss clinical risks associated with the infusion of a contaminated HSC product. Finally, we discuss the expectations for current good tissue practices (cGTP) and current good manufacturing practices (cGMP) for the manufacturing and testing of HSC based on Section 361 and Section 351 categorization, respectively. We provide commentary on what is practiced in the field and discuss the critical need for updates to professional standards that keep pace with advancing technologies with an aim to clarify expectations for manufacturing and testing facilities to improve standardization across institutions.

228 - First Reported Evaluation of Outcomes and Safety of Using “Flipped” Conditioning Regimens of Fractionated Total Body Irradiation Followed By Fludarabine Vs Cyclophosphamide for Allogeneic-Hematopoietic Stem Cell Transplant at the University of Southern California

228 - First Reported Evaluation of Outcomes and Safety of Using “Flipped” Conditioning Regimens of Fractionated Total Body Irradiation Followed By Fludarabine Vs Cyclophosphamide for Allogeneic-Hematopoietic Stem Cell Transplant at the University of Southern California

A Phase I Safety and Feasibility Study to Evaluate Motixafortide (CXCR4/SDF-1 Inhibition) and Natalizumab (VLA-4/VCAM-1 Inhibition) As a Novel Regimen to Mobilize CD34+ Hematopoietic Stem Cells for Gene Therapy in Sickle Cell Disease

Background: Sickle cell disease (SCD) is one of the most common genetic diseases globally. Clinical manifestations of SCD commonly include anemia, pain, vaso-occlusive events and cumulative end organ damage leading to significant morbidity and mortality. Historically, allogeneic hematopoietic cell transplantation (HCT) represented the only curative therapy for SCD, but is associated with significant HCT-related morbidity and mortality which limit its use (PMID: 8663884, PMID: 20007560). Autologous hematopoietic stem cell (HSC) -based gene therapies now offer curative potential with decreased toxicity (PMID: 28249145). However, effective HSC-based gene therapy depends on collection of sufficient HSCs (ideally >10x106 CD34+ cells/kg), typically from peripheral blood (PB) (PMID: 33956057). G-CSF and CXCR4 inhibition (CXCR4i) with plerixafor are the most widely used mobilization strategies (PMID: 19363221, PMID: 19720922). However, G-CSF is associated with risk of fatal vaso-occlusive events in SCD (PMID: 9734950, PMID: 11368061, PMID: 19513902). Meanwhile, short-acting CXCR4i with plerixafor alone does not reliably yield optimal HSC numbers for gene therapy applications (PMID: 30282642, PMID: 29419425, PMID: 29472357). Therefore, developing novel HSC mobilization regimens to rapidly and reliably mobilize optimal CD34+ HSCs for gene therapy in SCD represents an unmet need. Motixafortide (M) is a high-affinity, long-acting CXCR4 inhibitor. The recent Phase III, randomized, controlled GENESIS Trial demonstrated combination M+G-CSF resulted in 88.8% of patients collecting ≥6x106 CD34+ cells/kg (median 10.8x106) within 1 leukapheresis (LP) for HCT. This compares favorably to historical data with plerixafor+G-CSF where 54.2% collected to goal in 1 LP. Motixafortide also mobilized more immunophenotypically primitive HSCs and multi-potent progenitors compared to plerixafor. Meanwhile, transcriptional profiling by single-cell RNA sequencing revealed HSCs mobilized with M have gene expression profiles associated with enhanced regeneration and self-renewal, compared to G-CSF or plerixafor (DOI: 10.1182/blood-2021-144296). Motixafortide alone also mobilized an average of 11.6×106 CD34+ cells/kg in healthy volunteers, while mobilizing 91.6% of allogeneic HSC donors to goal in ≤2 LPs (median 6.07x106) (DOI: 10.1182/blood-2018-99-109701, PMID: 28835380). Natalizumab (N) is a monoclonal antibody to the a4 integrin subunit of a4b1 (VLA-4) capable of VLA4 inhibition (VLA4i). In mice and primates, N led to an ~8-fold increase in PB CD34+ HSCs in 24 hrs (PMID: 7692447). In humans, a single N infusion resulted in a 5- to 6-fold increase PB CD34+ HSCs. Meanwhile, the average PB CD34+ HSC levels after a single dose were similar to levels with repeated doses, indicating rapid mobilization of maximal PB HSCs by 24 hrs after N infusion (PMID: 18195093). Combination CXCR4i and VLA-4i has been well-tolerated and resulted in synergistic mobilization of HSCs in preclinical studies (PMID: 21886173). Therefore, we hypothesize that combination CXCR4i and VLA-4i with M+N represents a safe and effective strategy for robust, rapid and synergistic mobilization of HSCs to PB in patients with SCD. Study Design and Methods: This is a single center, single-arm, dose de-escalation, Phase 1 safety and feasibility study. Key eligibility criteria include patients with SCD (SS or Sβ0) age 18-35 years receiving automated RBC exchanges. Patients with prior HCT or gene therapy will be excluded. We estimate 5 patients in this pilot feasibility study will provide sufficient safety and preliminary efficacy data to guide further development of this novel mobilization strategy. All patients will have baseline labs prior to M (1.25mg/kg, subcutaneous), N (300mg, IV) and M+N dosing. Each patient will then undergo mobilization and LP (1 blood volume), per protocol (see Figure 1). The primary outcome will be to assess the safety and tolerability of M, N and M+N in each SCD patient, defined by dose-limiting toxicities and maximum tolerated dose. Key secondary outcomes include: kinetics of CD34+ HSC mobilization to PB (CD34+ cells/uL), number of CD34+ HSCs collected via LP (CD34+ cells/kg/L), incidence of AEs, pharmacokinetics and the recommended phase 2 dose. Correlative studies include immunophenotypic and single cell transcriptional profiling of CD34+ HSCs from LP product mobilized with M, N and M+N. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Outcomes of Myelofibrosis Patients in the Era of Using JAK2 Inhibitor with Allogeneic-HSCT at the University of Southern California

Introduction: In myelofibrosis (MF) patients, the standard of care curative option is with allogeneic-hematopoietic stem cell transplantation (allo-HSCT) but no clear standard of care pre- and post-allo-HSCT management exists. Questions then arise such as whether use of JAK inhibitors pre- or post-transplant lead to better outcomes. In addition, further elucidation of patient characteristics/risks/response are needed due to scarcity of data post-transplant. In this study, we aim to provide further data concerning this field. Methods: This retrospective chart review included patients aged 18 years with MF diagnosed in 2012-2020 and received allo-HSCT at our institution. Primary objectives include over-all survival (OS) and progression-free-survival (PFS) since transplant, stratified by risk, peri-transplant JAK2 inhibitor use and conditioning + associated graft versus host disease prophylaxis (GVHD ppx) use. Secondary objectives include patient/treatment/transplant characteristics, response, survival and GVHD rates. Two-sided p value ≤ 0.05 was significant. RESULTS: 21 screened MF patients received allo-HSCT at our institution, median age 61.1 years (85.7% non-Hispanic and 14.3% Hispanic). 71.4% had primary MF. 9.5% had unfavorable/high risk karyotype/cytogenetics with 38.1% at high risk per Dynamic International Prognostic Scoring System (DIPSS) Plus, and 38.1% high risk, 28.6% very high-risk per Mutation-Enhanced International Prognostic Score System (MIPSS) 70+ v2.0. 52.4% had JAK2 V617F mutation, 28.6% triple negative (lacked JAK2/MPL/CALR mutations) with 23.8% as having any positivity in either ASXL1, EZH2, SRSF2, IDH1/2 and U2AF1 mutations. Pre-transplant, 100% had pancytopenia requiring transfusions. 9.5% had CR/PR, 42.9% had PD and 47.6% had SD. Of note, 57.9% had any peri-transplant JAK2 inhibitor use (defined as use within 3 months pre- and post-transplant) with or without other MF related-treatments. 33.3% underwent MSD, 38.1% MUD and 28.6% underwent HAPLO allo-HSCT. 80% had reduced intensity conditioning (RIC) with an equal distribution of fludarabine-based regimens. In terms of GVHD ppx, 55% received tacrolimus+ methotrexate and 35% had post-transplant cyclophosphamide (PTCY). 30% underwent HAPLO + PTCY. In terms of outcomes and adverse events, pancytopenia improved from 90.5% at d+30 to 57.1% at d+365. Transfusion dependency decreased from 60% from d+90 to 26.7% by d+365. None had leukemic transformation. Clinical improvement rates increased from 33.3% d+30 to 78.6% by d+365. CR/PR rate increased from 5% at d+90 to 28.6% at d+365. 14.3% had relapsed/PD, 37.5% had primary graft failure with 33.3% any deaths post allo-HSCT. 42.9% had any acute GVHD pre-d+100, 42.1% had any chronic GVHD post d+100. Of note, 100% of our population were alive at d+100, and 78.9% alive by d+365. Since transplant, 3y OS was 62.6%, 3y EFS at 59.2% with corresponding worsening of OS/EFS with higher DIPSS (p=0.19-0.33) and a 3y OS (40%) (p=0.29) and EFS (40%) (p=0.41) in patients with very-high risk per MIPSS 70+ v2.0. With peri-transplant JAK2 inhibitor use, 3y OS was 56.8% vs 75% without; 3y EFS was 51.1% vs 75% without (p=0.33-0.51) (Fig.1). For patients who underwent HAPLO+PTCY GVHD ppx, OS and EFS reached at 16 months while patients who underwent non-HAPLO+ any GVHD ppx did not reach their median OS yet and achieved an EFS median of 57 months. 2y OS and EFS was at 50% for those who underwent HAPLO+PTCY vs a 67.1% OS and a 61.9% EFS for those who had non-HAPLO+ any GVHD ppx (p=0.51-0.72) (Fig.2). CONCLUSIONS: Given that most of our patients had high/very high-risk scores, we achieved a good rate of clinical improvement with less transfusion dependency. A good survival rate was also achieved (100% survival at d+100 and 78.9% at 1y). Our 3y OS of 62.6% and 3y EFS of 59.2% is also appreciable even with the rates of GVHD. Our data also further supports that having a higher risk score per DIPSS/MIPSS leads to poorer survival. Interestingly, there may be no difference in OS/EFS with peri-transplant JAK2 inhibitor use. Also, those who underwent HAPLO+PTCY had their median OS/EFS reached first (16 months) with a possible lower survival trend compared to those who underwent MSD/MUD + any GVHD ppx combination treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

First Reported Evaluation of Outcomes and Safety of Using "Flipped" Conditioning Regimens of Fractionated Total Body Irradiation Followed By Fludarabine Vs Cyclophosphamide for Allogeneic-Hematopoietic Stem Cell Transplant at the University of Southern California

First Reported Evaluation of Outcomes and Safety of Using "Flipped" Conditioning Regimens of Fractionated Total Body Irradiation Followed By Fludarabine Vs Cyclophosphamide for Allogeneic-Hematopoietic Stem Cell Transplant at the University of Southern California

CT-132 Outcome of Sex-Mismatched Allogeneic Hemopoietic Stem Cell Transplantation from Human Leukocyte Antigen–Identical Sibling Donors in Patients With Hematologic Diseases

Hematopoietic stem cell transplantation is a procedure aiming to cure hematologic diseases. However, graft-versus-host disease (GVHD) and disease relapse/failure are common side effects of this treatment. Sex-mismatched transplantation, particularly with female donors and male patients, is known to be associated with higher incidence of GVHD or lower incidence of survival. This is caused by the antibodies against minor histocompatibility antigens found on the Y chromosome of the male recipient, which were found to be associated with increased risk of chronic GVHD and non-relapse mortality. The study aimed to determine the incidence of acute and chronic GVHD, overall survival, and non-relapse mortality when male patients received stem cells from female matched sibling donors in allogeneic hematopoietic stem cell transplantation. Retrospective study over 8 years. Nasser Institute Hospital. In this study, patients were divided into 2 groups. Group 1: female donors to male patients (245 patients). Group 2: male donors to female patients (186 patients) and same-sex donors and patients (506 patients). The median age was 20 years (7 months-60 years). The outcome of transplants in the female-to-male group was compared with that of the male-to-female and same-sex group. Overall survival, incidence of acute and chronic GVHD, non-relapse mortality, and relapse/failure rate. Overall survival was 68.6% in group 1 compared to 69.5% in group 2 (P=0.5). Acute GVHD was 24% in group 1 compared to 19.6% in group 2 (P=0.1). Chronic GVHD was 15.1% in group 1 compared to 9.6% in group 2 (P=0.02). Non-relapse mortality was 27.7% in group 1 compared to 25.7% in group 2 (P=0.5). Relapse/failure rate was 4.9% in group 1 compared to 6.5% in group 2 (P=0.3). Allogeneic stem cell transplantation for male patients from female donors can be associated with a significantly higher rate of chronic GVHD but a lower incidence of relapse/failure.