Robust Engraftment Research Articles

Feasibility of Human Neural Stem Cell Transplantation for the Treatment of Acute Subdural Hematoma in a Rat Model: A Pilot Study.

Human neural stem cells (hNSCs) transplantation in several brain injury models has established their therapeutic potential. However, the feasibility of hNSCs transplantation is still not clear for acute subdural hematoma (ASDH) brain injury that needs external decompression. Thus, the aim of this pilot study was to test feasibility using a rat ASDH decompression model with two clinically relevant transplantation methods. Two different methods, in situ stereotactic injection and hNSC-embedded matrix seating on the brain surface, were attempted. Athymic rats were randomized to uninjured or ASDH groups (F344/NJcl-rnu/rnu, n = 7–10/group). Animals in injury group were subjected to ASDH, and received decompressive craniectomy and 1-week after decompression surgery were transplanted with green fluorescent protein (GFP)-transduced hNSCs using one of two approaches. Histopathological examinations at 4 and 8 weeks showed that the GFP-positive hNSCs survived in injured brain tissue, extended neurite-like projections resembling neural dendrites. The in situ transplantation group had greater engraftment of hNSCs than matrix embedding approach. Immunohistochemistry with doublecortin, NeuN, and GFAP at 8 weeks after transplantation showed that transplanted hNSCs remained as immature neurons and did not differentiate toward to glial cell lines. Motor function was assessed with rotarod, compared to control group (n = 10). The latency to fall from the rotarod in hNSC in situ transplanted rats was significantly higher than in control rats (median, 113 s in hNSC vs. 69 s in control, P = 0.02). This study first demonstrates the robust engraftment of in situ transplanted hNSCs in a clinically-relevant ASDH decompression rat model. Further preclinical studies with longer study duration are warranted to verify the effectiveness of hNSC transplantation in amelioration of TBI induced deficits.

Robust Engraftment with Mgta-456, a CD34+ Expanded Cell Therapy Product in Patients with Inherited Metabolic Disorders (IMD): Preliminary Phase 2 Trial Results

Background Mucopolysaccharidosis type IH (MPS1/Hurler Syndrome), cerebral adrenoleukodystrophy (cALD), metachromatic leukodystrophy (MLD) and globoid cell leukodystrophy (GLD) are fatal IMDs affecting the central nervous system treatable through allogeneic hematopoietic stem cell transplantation (HSCT). In the absence of a non-carrier HLA matched related donor, cord blood (CB) is the preferred source of cells in IMDs due to rapid availability and flexibility in matching. However, prolonged neutropenia and high graft failure rates present a challenge, particularly with reduced intensity conditioning regimens. MGTA-456 is produced from a single CB unit using an aryl hydrocarbon receptor antagonist in a 15-day expansion culture of CD34+ cells. In previous phase 1/2 studies, hematologic malignancy patients treated with myeloablative conditioning and MGTA-456, with median 324-fold CD34+ expansion, showed 100% engraftment with a 9 day reduction in time to neutrophil recovery. As higher CD34+ doses have been correlated with improved engraftment in IMD transplant patients, we hypothesize MGTA-456 may reduce duration of neutropenia and risk of graft failure and more rapidly correct the metabolic deficiency in IMD patients. Patients and Methods A Phase 2, open-label trial (NCT03406962) initiated in Feb 2018 is enrolling up to 12 patients Results Five patients have been treated per protocol to date (Figure 1). MGTA-456 contained a median 561-fold expansion of CD34+ cells after culture with a median infused CD34+ cell dose of 110 × 106 cells/kg and median total nucleated cell dose of 26.4 × 107 (Figure 2). The median duration of neutropenia was 1 day (range 0-9), in contrast to a median of 8 days for a historical cohort (Figure 3). Myeloid chimerism was ≥98% donor by day +14 in evaluable patients. Two patients developed autoimmune cytopenia, a known complication in IMD patients after HSCT, unrelated to MGTA-456. Time to discharge after transplant was a median of 19.5 days at time of reporting. Preliminary data from MPS1 patients demonstrated normalization of blood alpha-L-iduronidase enzyme activity and decreased urinary glycosoaminoglycan levels after transplant. Conclusions Treatment of IMD patients with MGTA-456 showed early and robust engraftment in all patients with marked reduction in days of neutropenia compared to a historical cohort. These preliminary data, in combination with the previous hematologic malignancy patients treated, suggest MGTA-456 substantially enhances engraftment and rate of neutrophil recovery.

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.

Persistence of CRISPR/Cas9-Edited Hematopoietic Stem and Progenitor Cells and Reactivation of Fetal Hemoglobin in Nonhuman Primates

Beta-thalassemia and sickle cell disease are monogenic disorders that are currently treated by allogeneic bone marrow (BM) transplantation although the challenges of finding a suitable matched-donor and the risk of graft vs host disease have limited the adoption of this otherwise curative treatment. A potentially promising approach for hemoglobinopathies aims to reactivate fetal hemoglobin (HbF) as a substitute for defective or absent adult hemoglobin by modifying the patient's own hematopoietic stem and progenitor cells (HSPCs). Here, we evaluated CRISPR/Cas9-induced small deletions in HSPCs that are associated with hereditary persistence of fetal hemoglobin (HPFH) using our nonhuman primate (NHP) stem cell transplantation and gene therapy model.The CRISPR/Cas9 nuclease platform was employed to recapitulate a natural genetic alteration identified in individuals with HPFH, consisting of a 13-nucleotide (nt) deletion in the gamma globin gene promoter. A first cohort of three rhesus macaques received 70-75% HPFH-edited BM-derived CD34+ HSPCs. All animals showed rapid hematopoietic recovery and peripheral blood (PB) editing levels stabilized at 12-30% for at least a year post transplantation (Figure 1). HbF production, determined by circulating F-cells, persisted at frequencies of 8-22% and correlated with in vivo PB editing. Robust engraftment of gene-edited HSPCs in the BM compartment was observed in all animals, with no measurable off-target activity or clonal expansion.We have recently shown, that the CD34+CD90+CD45RA- phenotype is exclusively required for short- and long-term multilineage reconstitution, significantly reduces the target cell number for gene therapy/editing and is conserved between human and NHP hematopoietic cells (Radtke et al., STM, 2017). To explore this cell population further, we transplanted a second cohort of three animals by sort-purifying and solely editing this hematopoietic stem cell (HSC)-enriched CD34+CD90+CD45RA- phenotype, thus reducing the number of target cells by over 10-fold without impacting hematopoietic recovery, engraftment, or HbF reactivation. In vivo levels of gene-edited PB started at less than 5% because of the high number of co-infused unmodified progenitor cells, but rapidly increased to about 50% within 1 week (Figure 1) and stabilized at levels comparable to the CD34 cohort. This data supports our interpretation that CD34+CD90+CD45RA- cells are the main cell population relevant for long-term reconstitution and an excellent target for improved and efficient gene therapy/editing.These results demonstrate robust engraftment and persistence of CD34+ HPSCs as well as HSC-enriched CD34+CD90+CD45RA- cells that have been CRISPR/Cas9-edited at the 13nt-HPFH site, with marked and stable HbF reactivation and no overt adverse effects in a NHP transplantation and gene therapy model. Most importantly, we validated our refined CD90+ target which reduces the need for editing reagents by 90% without compromising the gene modification and engraftment efficiencies. These are the first data in a clinically relevant large animal model to demonstrate the feasibility and clinical applicability of CRISPR/Cas9-mediated fetal hemoglobin reactivation. The successful targeting and engraftment of our HSC-enriched population should also have significant implications for gene therapy and editing of other genetic diseases.Figure 1:Tracking of HPFH editing in transplanted animals. A) Editing efficiency was longitudinally determined by next generation sequencing of the targeted locus in PB white blood cells from 2 cohorts of transplanted rhesus animals. Frequency is represented as the proportion of all sequence reads containing an edited locus. B) Normalized frequency of the desired 13nt-HPFH deletion in the same animals as shown in A). DisclosuresNegre:Bluebird Bio: Employment, Equity Ownership, Other: Salary. Adair:RX Partners: Honoraria; Miltenyi Biotec: Honoraria; Rocket Pharmaceuticals: Patents & Royalties: PCT/US2017/037967 and PCT/US2018/029983. Scharenberg:Generation Bio: Equity Ownership; Casebia Therapeutics: Employment; Alpine Immune Sciences: Equity Ownership. Kiem:Rocket Pharmaceuticals: Consultancy; Magenta: Consultancy; Homology Medicine: Consultancy.

Preliminary Phase 2 Results Demonstrate Engraftment with Minimal Neutropenia with MGTA-456, a CD34+ Expanded Cord Blood (CB) Product in Patients Transplanted for Inherited Metabolic Disorders (IMD)

▪Background: IMDs including mucopolysaccharidosis type IH (MPS1/Hurler Syndrome), metachromatic leukodystrophy (MLD), globoid cell leukodystrophy (GLD) and cerebral adrenoleukodystrophy (cALD) are progressive, fatal diseases affecting the central nervous system which are treatable through allogeneic hematopoietic stem cell transplantation (HSCT). CB, in the absence of a matched donor, is the preferred source of stem cells as it is rapidly available and allows greater flexibility in allele matching. As a result of low cell doses, CB transplants in IMD are associated with prolonged periods of neutropenia and reported graft failure rates in up to ~20% (Lum et al 2017 Bone Marrow Transplant 52:846-53; Mallhi et al 2017 BBMT 23:119-25). MGTA-456 is a first-in-class cell therapy produced from a single CB unit using an aryl hydrocarbon receptor antagonist in a 15-day expansion culture of CD34+ cells. In previous phase 1/2 studies, 24 adult and 3 pediatric patients with hematologic malignancies treated with myeloablative conditioning (MAC) and MGTA-456 demonstrated a median 324-fold expansion of CD34+ cells, all patients engrafted, and the time to neutrophil recovery was significantly reduced by a median of 9 days compared to historical controls (Wagner et al 2016 Cell Stem Cell 18:144-55; Wagner et al 2017 Blood 130 supp:662 abstr). Furthermore, higher CD34+ dose has been correlated with improved engraftment and outcomes in IMD transplant patients (Prasad et al 2008 Blood 112:2979-89). Based on these promising data, we postulate the increased CD34+ dose provided by MGTA-456 would reduce the length of neutropenia and risk of graft failure in IMD patients.Patients and Methods: A Phase 2, open-label trial (NCT03406962) initiated in Feb 2018 is enrolling up to ~12 patients <16 yo with a diagnosis of MPS1, cALD, MLD, and GLD lacking a non-carrier matched related donor. Eligible CB units were matched at ≥ 6 of 8 HLA loci (A, B, C and DRB1) using allele-based typing. The reduced toxicity MAC regimen consists of anti-thymocyte globulin (days -9 to -6) followed by fludarabine (40 mg/m2 days -5 to -2) and busulfan (total exposure 21,000 to 22,000 μM/min/L-1 days -5 to -2). Immunoprophylaxis consists of cyclosporin and methylprednisolone.Results: Four patients have been treated thus far (Table 1). The MGTA-456 process provided a marked expansion of CD34+ cells (median 482-fold, Figure 1) with a median infused CD34+ cell dose of 84 x 106 cells/kg and median total nucleated cell (TNC) dose from the expanded fraction of 19.6 x 107 (Table 1). TNC dosing was capped at 27.0 x 107 cells/kg per protocol. The only infusion-related reaction noted was grade 3 nausea in 1 patient. Two patients had no days of neutropenia and 2 patients had 1 and 4 days respectively (mean 1.25 days) in contrast to a mean of 7.8 days for a historical cohort of 27 IMD patients undergoing CB transplantation at the same institution with identical conditioning (Figure 2). Myeloid chimerism (CD33+/66+) achieved ≥98% donor by day +14 in all patients. For the 3 patients with data at time of reporting, days to discharge after transplant were 17, 12 and 18. No patients experienced acute or chronic GVHD. One patient developed autoimmune cytopenia (not related to MGTA-456) which is a known complication reported in 20-56% of IMD patients undergoing HSCT that resulted in death at day +143 (Page et al 2008 BBMT 14:1108-17; Khalil et al 2014 Sci World J 581657). Long-term disease specific outcome measures including enzymatic activity are being collected and will be reported over time.Conclusions: Preliminary results of transplantation in IMD patients with MGTA-456, containing highly expanded CD34+ cell doses, demonstrated early and robust engraftment in all patients with marked reduction in days of neutropenia (to 0-4 days or mean 1.25 days) in comparison to a historical cohort. MGTA-456 was well tolerated with one infusion-related event of grade 3 nausea. These data, in combination with the previous 27 hematologic malignancy patients treated, suggest that MGTA-456 substantially enhances the engraftment potential of CB. Based on these promising data, MGTA-456 has potential to improve transplant-related outcomes in patients undergoing HSCT and increasing the availability of well-matched CB units that may have been previously excluded due to inadequate CD34+ dose. [Display omitted] DisclosuresOrchard:Magenta Therapeutics: Research Funding. Raffel:Magenta Therapeutics: Employment, Equity Ownership. Condon:Magenta Therapeutics: Employment, Equity Ownership. Monaghan:Magenta Therapeutics: Employment, Equity Ownership. Vernet:Magenta Therapeutics: Employment. Sheirr:Magenta Therapeutics: Employment, Equity Ownership. Braun:Magenta Therapeutics: Research Funding. Shanley:Magenta Therapeutics: Research Funding. Lund:Magenta Therapeutics: Research Funding. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Davis:Magenta Therapeutics: Employment, Equity Ownership. Wagner:Novartis: Research Funding; Magenta Therapeutics: Consultancy, Research Funding.

Conditioning for Hematopoietic Stem Cell Transplantation Using Antibody-Drug Conjugate Targeting CD45 Permits Engraftment across Immunologic Barriers

INTRODUCTION: Hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for benign and malignant hematologic diseases. It also holds great promise as treatment for infectious and autoimmune disease and for autologous gene therapy. However, due to the toxicity of conditioning regimens for HSCT, it is generally reserved for the most life-threatening diagnoses, such as acute leukemia. Targeted approaches to conditioning may improve the safety of HSCT and extend its applicability to other diseases. Recently, conditioning regimens were described consisting of treatment with anti-CD45.2 or anti-cKit antibodies conjugated to the ribosome-inhibiting protein saporin (CD45-SAP and cKit-SAP, respectively) (Palchaudhuri et al (2016), Nat Biotechnol, Czechowicz et al (2016), ASH Abstract). These were reported to deplete murine hematopoietic stem cells (HSCs) and permit engraftment of syngeneic HSCs with minimal toxicity. However, the ability of CD45-SAP and cKit-SAP to permit HSCT across immunologic barriers without chemotherapy or radiation, or to target malignant cells expressing these receptors, are potential applications that were not explored.METHODS: We compared CD45-SAP and cKit-SAP conditioning in syngeneic (B6 to B6) and allogeneic (CB6F1 to B6) murine transplantation models. Donor chimerism in peripheral blood (PB) and complete blood counts (CBCs) were assessed monthly during these experiments. Successful HSC engraftment was confirmed by serial transplantation experiments. Human CD45-SAP and cKit-SAP conjugates were also tested for their ability to target MOLM13 AML cells using XTT viability assays, and in targeting cord-blood derived HSCs using colony forming cell assays.RESULTS: We confirmed that CD45-SAP dosed at 75 µg per mouse could effectively deplete B6 mice of HSCs and condition them for syngeneic HSCT as reported (Palchaudhuri et al (2016), Nat Biotechnol), with normal-range CBCs and stable donor PB chimerism of approximately 80% at 6 months post-transplant. Donor-derived HSC in CD45-SAP treated mice successfully engrafted upon transplantation to secondary B6 recipients. cKit-SAP dosed at 10 µg per mouse was sufficient for HSC depletion, but robust syngeneic engraftment required dosing at 50 µg per mouse. Notably, CD45-SAP treatment combined with CD4+and CD8+ T cell depletion permitted haploidentical transplantation from CB6F1 donors to B6 recipients, with normal-range CBCs and stable donor PB chimerism of approximately 70% at 3 months post-transplant. Finally, human CD45-SAP conjugates could successfully target MOLM13 leukemia cells and cord-blood derived HSCs in vitro.CONCLUSIONS: Transplantation in mice across immunological barriers is achievable using CD45-SAP conditioning, with high-level donor chimerism without chemotherapy or radiation. Furthermore, human CD45-SAP conjugates can successfully target human leukemia cell lines and primary HSCs in vitro. Further studies are underway in three areas. First, by combining T and NK cell depletion with CD45-SAP conditioning, we are attempting full-mismatch HSCT in mice. Second, using humanized mouse models, we will assess the ability of CD45-SAP to target human leukemia cells and HSCs in vivo. Finally, we are exploring using HSCT with CD45-SAP conditioning as a treatment in mouse models of autoimmune disease. DisclosuresRettig:Amphivena Therapeutics: Research Funding; Novimmune: Research Funding.

Non-Genotoxic Conditioning Efficiently Depletes Host Hematopoietic Stem Cells and Facilitates Robust Multi-Lineage Engraftment in a Mouse Model of Fanconi Anemia

Conditioning chemotherapy is used to deplete hematopoietic stem cells in the recipient's marrow prior to a bone marrow transplant to facilitate engraftment of donor cells. While effective, some major issues with chemotherapy remain which includes off-target genotoxic effects increasing the risk of secondary malignancies. These complications are compounded in disease settings that arise from a deficit in DNA repair pathways where cytotoxic treatments can result in oncogenic transformation, such as Fanconi anemia (FA). FA is an inherited bone marrow failure disorder resulting from an intrinsic defect in DNA repair affecting approximately 130,000 children each year. Currently mutations in 22 genes have been implicated in the pathogenesis of FA. While novel gene-therapy based protocols are showing early promise for this patient populations, the standard treatment for the hematologic complications for FA is a bone marrow transplant. However, secondarily to an underlying sensitivity to DNA damage, these patients are unable to receive standard myeloablative conditioning, which can reduce the efficiency of reconstitution. Avoiding alkylating agents could improve outcomes and success rates in these patients. Furthermore, this methodology could be translated to the allogeneic bone marrow transplantation setting, decreasing the toxicity of this treatment modality.Our approach for characterizing an alternative conditioning regimen for FA patients utilizes immunotoxin conjugates specifically targeting hematopoietic stem cell populations. These non-genotoxic antibody-based drug conjugates utilize saporin (SAP), a ribosomal toxin, to eliminate cells in a targeted manner while leaving the remainder of the marrow compartment intact. Antibodies targeting either CD45 or CD117 were used in a mouse model of FA where expression of their FANCA gene, one of the most common mutations in humans, has been knocked out. Mice conditioned with either of the drugs received various doses of whole marrow from healthy heterozygous littermates. On the day of transplant, mice conditioned with either immunotoxin demonstrated significantly reduced LSK stem cell populations in the marrow similar to cyclophosphamide (Cy) controls (Figure 1A).Peripheral engraftment of donor cells was monitored for 6 months, after which mice were sacrificed for complete analysis of engraftment in the bone marrow compartment. No significant difference was observed in engraftment between Cy and immunotoxin conditioned mice (Figure 1B), and all treatment groups exhibited robust multilineage reconstitution in a cell dose dependent manner. Additionally, Cy treated mice demonstrated greater and sustained weight loss and lower gastrointestinal losses compared to immunotoxin treated mice. Ongoing clonal analysis studies of engrafted cells has demonstrated polyclonal reconstitution, indicating a large number of donor stem cells are actively contributing to hematopoiesis.In this study, we demonstrate that non-genotoxic conditioning approaches both facilitate multilineage engraftment of donor marrow and significantly deplete host hematopoietic stem cell populations. These are crucial since persistence of host hematopoiesis could eventually result in clonal evolution and leukemogenesis in post-transplant FA patients. Achieving both of these conditions through targeted elimination with immunotoxin conjugates represents a major advancement in bone marrow transplantation for FA. We now are initiating studies using immunotoxin-based conditioning for the transplantation of gene-modified syngeneic stem cells and allogeneic cells. We think these studies will inform future clinical trials and provide the groundwork for the next-generation of therapy for FA patients. [Display omitted] DisclosuresHartigan:Magenta Therapeutics: Employment. Palchaudhuri:Harvard University: Patents & Royalties; Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Kiem:Homology Medicine: Consultancy; Magenta: Consultancy; Rocket Pharmaceuticals: Consultancy.

Ex Vivo Gene-Edited Cell Therapy for Sickle Cell Disease: Disruption of the BCL11A Erythroid Enhancer with Zinc Finger Nucleases Increases Fetal Hemoglobin in Plerixafor Mobilized Human CD34+ Cells

Increases in fetal hemoglobin (HbF) are associated with improved clinical outcomes in the inherited hemoglobinopathies. We are developing a novel gene-edited cell therapy to treat patients with sickle cell disease (SCD) and beta-thalassemia (BT) using autologous hematopoietic stem and progenitor cells (HSPCs) genetically modified with zinc finger nucleases (ZFNs) to restore high levels of HbF expression. The ZFNs have been designed to specifically target the GATA motif within an intronic erythroid-specific enhancer (ESE) of BCL11A, the gene encoding a transcriptional regulator of the fetal-to-adult hemoglobin switch. Previously, we reported successful ZFN-mediated, ex vivoBCL11A gene editing in dual mobilized HSPCs, i.e. peripheral stem cells mobilized with a combination of granulocyte colony-stimulating factor (G-CSF) and plerixafor1. The editing procedure was optimized for high on-target/low off-target modification levels and increases in HbF in erythroid progeny. This drug product, ST-400, passed extensive safety testing and is currently in a phase 1/2a clinical trial for transfusion-dependent beta-thalassemia (ClinicalTrials.gov number NCT03432364).An analogous drug product, BIVV003, is being developed as a therapeutic for SCD. In patients with SCD, the use of G-CSF for stem cell mobilization is not recommended due to the risk of clinical complications. Therefore, peripheral HSPCs are obtained from SCD patients via single agent, plerixafor mobilization and apheresis2-5. Understanding the effect of this change in mobilization strategy on ZFN editing efficiency and specificity is a key element in preparing for SCD gene-edited cell therapy. In the present studies, we demonstrate comparability of ZFN editing outcomes in single and dual mobilized HSPCs obtained from healthy donors. In plerixafor mobilized HSPCs from five healthy donors at research scale, ZFN-mediated gene editing induced an efficient modification at the BCL11A ESE target site (>75% of alleles modified, as measured by MiSeq deep sequencing) with high post-editing viability (77%). Similar gene editing efficiencies (>70%) were obtained in HSPCs at clinical manufacturing scale (n=2). Further, in vitro HbF protein levels and HbF+ cell frequencies within erythroid progeny of edited cells were increased by >4 and 3-fold respectively - compared to non-edited cells in the same culture conditions, using reverse phase high-performance liquid chromatography and flow cytometry (n=4 healthy donors at research scale). Single cell clone analysis revealed that ZFN-mediated gene editing targeted both alleles of BCL11A at high frequency (91-94% of edited cells within erythroid progeny) with high levels of replicable GATA-disrupting indel patterns. On average, each edited allele contributed an additional 17.6% increase in HbF production in vitro, with a statistically-significant increase in HbF level for biallelic edited vs. unedited controls (3.4 fold).Critical to BIVV003 use in clinical trials, ZFN-mediated gene editing did not impair single agent mobilized HSPC function in vitro based on measurements of colony forming unit (CFU) production and frequencies of long-term HSC (LT-HSC) and common myeloid progenitor (CMP) cells by flow cytometry. In agreement with this data, injection of BIVV003 into immune-deficient NBSGW mice resulted in robust long-term engraftment (21 weeks) without any impact on the number of HSPCs and their differentiated progeny. Overall, these data demonstrate potential efficacy of ZFN-edited HSPCs (BIVV003) as a novel cell therapy for SCD patients.Holmes et al., 2017 (ASH abstract)Fitzhugh et al., 2009Lagresle-Peyrou et al., 2018Hsieh and Tisdale, 2018Yannaki et al., 2012 DisclosuresMoran:Bioverativ, a Sanofi Company: Employment. Ling:Bioverativ, a Sanofi Company: Employment. Lessard:Bioverativ, a Sanofi Company: Employment. Vieira:Bioverativ a Sanofi Company: Employment. Hong:Bioverativ, a Sanofi Company: Employment. Holmes:Sangamo Therapeutics: Employment. Reik:Sangamo Therapeutics: Employment. Dang:Sangamo Therapeutics: Employment. Gray:Sangamo Therapeutics: Employment. Levasseur:Bioverativ, a Sanofi Company: Employment. Rimmele:Bioverativ, a Sanofi Company: Employment.

Engineering Resistance to CD33-Targeted Immunotherapy in Normal Hematopoiesis By CRISPR/Cas9-Deletion of CD33 Exon 2

Background:Improved survival with gemtuzumab ozogamicin (GO) in some people with acute myeloid leukemia has validated CD33 as immunotherapeutic target and sparked interested in developing new, highly potent CD33-directed therapeutics. As a limitation of this treatment strategy, CD33 expression on maturing and mature myeloid cells causes significant on-target, off-leukemia effects. Toxicity of CD33-targeted immunotherapy should be minimal in the presence of normal hematopoietic stem and progenitor cells (HSPCs) engineered to lack CD33 variants recognized by therapeutic antibodies. Indeed, very recent studies have shown that CRISPR/Cas9 with a single guide RNA (gRNA) designed to target the CD33coding region reduces display of CD33 and protects engineered cells from CD33 CAR T-cells. However, this approach resulted in low levels of CD33disruption in vivoand off-target activity. We therefore developed an approach in which the Cas9 protein is complexed with two synthetic gRNAs for precise excision of the intervening sequence (i.e. more controlled genome editing than what can be accomplished with a single gRNA). We directed these two gRNAs to intronic sequences for precise excision of exon 2, which encodes the V-set domain of CD33 that is recognized by all current CD33 therapeutics including GO. This approach eliminates exonic indels and protects engineered cells from CD33-targeted immunotherapy while maintaining expression of an exon 2-free variant of CD33 (CD33∆E2), which has previously been identified as natural isoform in human HSPCs.Methods: We used human myeloid ML-1 cells and human fetal liver CD34+ HSPCs for CRISPR/Cas9 editing, which was carried out by electroporation of purified Cas9 protein complexed with synthetic gRNAs. In vitro cytotoxicity assays were performed to test sensitivity to GO and the CD33/CD3 bispecific antibody AMG 330.For in vivoassessment of engineered HSPCs, NSG neonate mice were infused with 6.0x105human CD34+ cells, and peripheral blood analyzed biweekly for 14 weeks.Results:Delivery of Cas9/sgRNA ribonucleoproteins (RNPs) in ML-1 cells resulted in exon 2 deletion and time-dependent reduction in cell surface display of full-length CD33 (CD33FL). Pure populations of CD33∆E2cells were generated via single-cell cloning. These sublines lacked surface display of CD33FLbut, instead, expressed increased levels of the CD33∆E2transcript. CD33∆E2ML-1 cells were completely resistant to GO and AMG 330, whereas wild-type ML-1 cells were highly sensitive to these two drugs. Consistent with the findings in ML-1 cells, delivery of CRISPR/Cas9 RNPs to human fetal liver CD34+ HSPCs resulted in the expected CD33exon 2 deletion and decreased CD33FLsurface expression, while it did not impact the distribution of colony-forming cellsand only minimally reduced colony-forming potential. In NSG mouse xenotransplantation experiments, we found these CD33∆E2human CD34+ HSPCs to have comparable engraftment and multilineage differentiation potential relative to HSPCs expressing CD33FL(Fig. 1A). As expected, CD33FLexpression was substantially reduced in peripheral blood monocytes from CD33∆E2 HSPC-engrafted animals (Fig. 1B). In addition, robust engraftment of CD33∆E2 -engineered HSPCs in bone marrow was demonstrated by over 50% biallelic CD33exon 2 deletion at time of necropsy from colony-forming cells analysis (Fig. 1C). To determine if in vivodifferentiated myeloid CD33∆E2cells derived from infused human CD34+ HSPCs are indeed resistant to CD33-directed therapies, we treated mice with GO intravenously. GO administration reduced the number of circulating CD33FLCD14+ myeloid cells while leaving the number of CD33∆E2cells unaffected.Conclusion:These findings support a novel strategy in which CD33∆E2-engineeredHSPCs are used to widen the therapeutic window of CD33-directed immunotherapies. Such cells could be envisioned for people at risk for the need of CD33-immunotherapy, e.g. AML patients in remission (where CD33-immunotherapy could be used to prevent/treat relapse) or, pre-emptively, for people with genetic AML-predisposition syndromes (such that CD33-targeted immunotherapy could be given safely if AML occurred). With this potential, further development of CD33∆E2-engineered HSPCs toward clinical application is warranted. [Display omitted] DisclosuresWalter:Amphivena Therapeutics, Inc: Consultancy, Other: Clinical Trial Support, Research Funding; Aptevo Therapeutics, Inc: Consultancy, Other: Clinical Trial Support, Research Funding; Covagen AG: Consultancy, Other: Clinical Trial Support, Research Funding; Actinium Pharmaceuticals, Inc: Other: Clinical Trial support , Research Funding; Amgen Inc: Other: Clinical Trial Support, Research Funding; Boehringer Ingelheim Pharma GmbH & Co. KG: Consultancy; Pfizer, Inc: Consultancy; Seattle Genetics, Inc: Consultancy, Other: Clinical Trial Support, Research Funding. Kiem:Magenta: Consultancy; Homology Medicine: Consultancy; Rocket Pharmaceuticals: Consultancy.

High-Risk Langerhans Cell Histiocytosis in Infants Exhibits Malignant Features in a Xenograft Model but Responds Durably to Targeted Therapy

Background: Langerhans cell histiocytosis (LCH) is a rare hematologic neoplasm primarily of childhood, characterized by a neoplastic proliferation of Langerhans-like cells. The clinical presentation of LCH is highly variable, and while some children are cured with combination chemotherapy, many will relapse and experience irreversible morbidity. Genomic profiling of LCH has identified recurrent somatic activating mutations in BRAF and MAP2K1, all of which culminate in activation of the mitogen-activated protein kinase pathway. However, key mechanistic and clinical questions such as the curative potential of targeted therapy and the cell of origin remain unanswered.Methods: Four infants with risk-organ positive (RO+) multisystem BRAF V600E-mutant LCH and secondary hemophagocytosis were treated at our institution with the BRAF V600E-inhibitor dabrafenib after failing chemotherapy, or up front at the discretion of the treating physician. Response assessment was performed as indicated for good clinical care. Excess bone marrow obtained during routine clinical evaluation was collected under an IRB-approved protocol, and transplanted into busulfan-conditioned NOD/LtSz-SCID interleukin-2(IL2)RG-/- (NSG) or NSG-SGM3 (NSGS) mice. Donor chimerism was assessed by flow cytometry for mouse and human CD45 and lineage reconstitution was determined with standard human markers. BRAF V600E detection was performed on extracted DNA from xenografts using a TaqMan Mutation Detection RTPCR assay.Results: At presentation, all patients exhibited features of HLH (hemophagocytic lymphohistiocytosis) with fever, splenomegaly, pancytopenia, elevated markers of inflammation, hypofibrinogenemia, and hemophagocytosis on bone marrow examination. All patients displayed marked and rapid improvement following initiation of dabrafenib. With a median follow up of 21 months, all patients remain in complete clinical remission, and are thriving. None of the patients experienced significant adverse effects classically associated with dabrafenib. In two patients, despite clinical disease resolution, BRAF V600E was still detectable in bone marrow by RTPCR at 23 and 26 months respectively post initiation of dabrafenib. Remarkably, the other two patients who were treated upfront with dabrafenib achieved molecular remissions (defined as undetectable BRAF V600E allele in a previously positive patient) after 11 and 12 months respectively of therapy.Among mice transplanted with patient bone marrow cells, the median survival was 9.6 weeks. A comparison arm consisting of mice transplanted with bone marrow from a low-risk LCH patient did not develop evidence of disease during the observation period. At necropsy, animals exhibited splenomegaly, thrombocytopenia and anemia. Flow cytometry of both bone marrow and spleens from mice demonstrated robust engraftment of human hematopoietic cells, and the BRAF V600E allele was detected from marrow and splenocytes in all mice. Frequent hemophagocytic forms were visible on bone marrow cytospins, and on histologic examination, bone marrow exhibited an abundance of CD1a-negative, CD163+ histiocytes, similar to findings observed in human disease.Bone marrow and splenocytes from primary NSG xenograft recipients were transplanted into secondary NSGS mice, and two out of three animals examined similarly became ill with comparable disease latency, and achieved engraftment of human cells with detectable BRAF V600E in bone marrow and splenocytes.Conclusions: For patients with LCH with hematologic involvement and secondary HLH, disease-initiating cells are present in the bone marrow and can engraft and recapitulate disease following primary and secondary transplant into immunodeficient mice. Targeted therapy with the BRAF V600E-specific inhibitor dabrafenib leads to durable, sustained remissions. Furthermore, we demonstrate that certain patients can achieve molecular remission with BRAF V600E-specific monotherapy alone. However, in at least a subset of patients with RO+ LCH, the persistence of mutated BRAF despite prolonged targeted therapy suggests that while quiescent, transformed disease-initiating cells are not eliminated by BRAF inhibition alone. Based on these results, we hypothesize that first-line therapy with BRAF inhibitors may be more likely to induce molecular remissions. DisclosuresNo relevant conflicts of interest to declare.

Eltrombopag for Prolonged Post-Transplant Hyporigenerative Cytopenias

While vast majority of patients achieve robust engraftment within a few weeks after allogeneic hematopoietic stem cell transplantation (HSCT), some patients may experience persistent cytopenias, mostly thrombocytopenia, which are usually referred to as poor graft function (PGF). Unfortunately, treatment options for PGF remain limited, because except the possible treatment of the cause (which is often missing, and not always treatable) the only therapy is a boost of HSCT from the same donor. Eltrombopag is a thrombopoietin-mimetic agent which is approved for the treatment of idiopathic thrombocytopenia; more recently, it has been shown effective for the treatment of aplastic anemia. Between January 2011 and April 2018, 66 patients suffering from different malignant or non-malignant hematological diseases underwent an HSCT at the Federico II University of Naples. We report on a retrospective analysis of 13 transplanted patients who have consistently received eltrombopag for the treatment of post-HSCT persistent cytopenia. Patients were selected according the following criteria: lack of neutrophil engraftment at day +28 (primary graft failure), or persistent unilineage or multilineage cytopenia at anytime after HSCT (PGF; demonstration of impaired hematopoiesis by bone marrow analysis). All but one patients (who had severe aplastic anemia) were transplanted for a malignant disease, after a fully myelo-ablative conditioning regimen. There was a female prevalence (9 out 13); the median Sorror comorbidity index was 0 (range 0-2) while the median EBMT score was 4 (range 2-7). The most common HSC source was bone marrow (BM) (10 out 13 patients, 83,3%), and the median dose of CD34+ was 2.24 x 106/kg (range 0.68-7.2), with 4 out of the 13 patients (33%) who have received <2 x 106 CD34+/kg (but only 1 <1.5 x 106 CD34+/kg). Major ABO-incompatibility was present in 4/13 cases (25%); these patients underwent 2-3 sessions of plasmapheresis before receiving an unmanipulated BM. The vast majority of patients (9 out 13, 75%) were transplanted from a haploidentical donor, while 2 and 2 patient were grafted from unrelated or sibling HLA-matched donor. Considering patients evaluable for engraftment (alive at day +35, n=52) the incidence of PGF was not increased in haplo-identical HSCT (Fisher two-tailed exact test, p=0.32). All but one patients had ANC engraftment (median 18 days, range 14-34), with donor engraftment confirmed by full donor chimerism on bulk BM specimen. The patient suffering from primary graft failure underwent a second HSCT from the same donor, eventually dying from acute GvHD. Indeed, 11/12 patients were classified as having PGF based on low platelet counts; 6 of them were considered primary PGF because they have never reached platelets >20000/µL after HSCT. In most patients, PGF was not limited to the megakaryocytic lineage, since anemia and neutropenia affected respectively 6 and 2 patients. At the time of PGF, 6/12 patients had grade II-IV acute GvHD. Among the 12 patients with PGF, 5 patients achieved a complete response and 2 patient a partial response; among the 5 non-responders, 2 suffered from early deaths due to infectious complications and were not evaluable for response. Among the 6 responders, 3 of them have discontinued eltrombopag, while retaining their hematological response. The long-term outcome of this cohort was good, since only 3 deaths were observed (the 2 infections and one grade IV aGvHD). No patient experienced relapse of his underlying disease, eventually suggesting that eltrombopag should not preferentially stimulate possible residual malignant cells. In conclusion, our study suggest that eltrombopag is safe and can be hypothesized as etiologic treatment of PGF; indeed, eltrombopag may be considered in post-HSCT prolonged cytopenia due to PGF when other etiologic treatments are elusive or ineffective. Not all PGF share the same cause and pathogenic mechanism; in our preliminary experience eltrombopag seems more effective when additional specific causes can be identified (e.g., immune mechanism associated with GvHD, or infectious complications and their treatments). Future studies are needed to better understand the pathogenic events underlying PGF in individual patients, and to identify the setting of PGF patients who may best benefit from eltrombopag treatment. DisclosuresPane:BMS: Speakers Bureau; AMGEN: Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Risitano:Amyndas Pharmaceuticals: Consultancy; Pfizer Inc.: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alnylam Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Ra Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.

Localized immune tolerance from FasL-functionalized PLG scaffolds

Intraportal allogeneic islet transplantation has been demonstrated as a potential therapy for type 1 diabetes (T1D). The placement of islets into the liver and chronic immunosuppression to control rejection are two major limitations of islet transplantation. We hypothesize that localized immunomodulation with a novel form of FasL chimeric with streptavidin, SA-FasL, can provide protection and long-term function of islets at an extrahepatic site in the absence of chronic immunosuppression. Allogeneic islets modified with biotin and engineered to transiently display SA-FasL on their surface showed sustained survival following transplantation on microporous scaffolds into the peritoneal fat in combination with a short course (15 days) of rapamycin treatment. The challenges with modifying islets for clinical translation motivated the modification of scaffolds with SA-FasL as an off-the-shelf product. Poly (lactide-co-glycolide) (PLG) was conjugated with biotin and fabricated into particles and subsequently formed into microporous scaffolds to allow for rapid and efficient conjugation with SA-FasL. Biotinylated particles and scaffolds efficiently bound SA-FasL and induced apoptosis in cells expressing Fas receptor (FasR). Scaffolds functionalized with SA-FasL were subsequently seeded with allogeneic islets and transplanted into the peritoneal fat under the short-course of rapamycin treatment. Scaffolds modified with SA-FasL had robust engraftment of the transplanted islets that restored normoglycemia for 200 days. Transplantation without rapamycin or without SA-FasL did not support long-term survival and function. This work demonstrates that scaffolds functionalized with SA-FasL support allogeneic islet engraftment and long-term survival and function in an extrahepatic site in the absence of chronic immunosuppression with significant potential for clinical translation.

Human neuroepithelial stem cell regional specificity enables spinal cord repair through a relay circuit

Traumatic spinal cord injury results in persistent disability due to disconnection of surviving neural elements. Neural stem cell transplantation has been proposed as a therapeutic option, but optimal cell type and mechanistic aspects remain poorly defined. Here, we describe robust engraftment into lesioned immunodeficient mice of human neuroepithelial stem cells derived from the developing spinal cord and maintained in self-renewing adherent conditions for long periods. Extensive elongation of both graft and host axons occurs. Improved functional recovery after transplantation depends on neural relay function through the grafted neurons, requires the matching of neural identity to the anatomical site of injury, and is accompanied by expression of specific marker proteins. Thus, human neuroepithelial stem cells may provide an anatomically specific relay function for spinal cord injury recovery.

3041 - Robust Autonomous Generation of Definitive Hematopoietic Stem Cells From Pre-Circulation Murine Embryos

The embryonic anatomical site of origin of the definitive Hematopoietic Stem and Progenitor Cells (HSPCs) is still under debate in the field. An intra-embryonic origin (IE) is well accepted. In contrast, a yolk sac (YS) contribution to adult hematopoiesis remains controversial. The same developmental origin (e.g. endothelial cells with a mesodermal ancestor) makes it difficult to identify specific markers to discern between an IE- vs. YS-origin using a lineage trace approach. This together with the highly migratory nature of blood cells and the inability of cells isolated from pre-circulatory embryos to robustly engraft in transplantation assays, even after ex vivo culture, has precluded scientists from properly answering these questions. We have established ex vivo culture conditions to allow for the derivation of robust multi-lineage engraftment activity from pre-circulation murine embryos. CD45.2 + embryos at the 2–7 somite pairs (s.p.) stage were dissected from their YS, cultured as explants in our optimized conditions and transplanted into lethally irradiated CD45.1 + /CD45.2 + recipients. Here, we observed multilineage reconstitution of the peripheral blood (PB) for >16 weeks post-transplant in 16/18 recipients. This engraftment activity was serially transplantable. In primary recipients, PB chimerism was >50% in 8/18 total recipients. This robust PB chimerism allowed us to rigorously interrogate the corresponding CD45.2 + YS (cultured under the same conditions). In these transplants, we observed PB engraftment in 0/18 recipients of cells isolated from cultured YS. These data suggest that either YS does not autonomously generate HSPCs or that it can not support their maturation ex vivo. Additionally, we found that the engraftment ability from the IE is confined to an emerging CD31+CD45+c-Kit+CD41- population. Importantly, this population also develops in the YS explants, but lacks engraftment ability. In sum, our work supports a model in which the embryo, not the YS, is the major source of lifelong hematopoiesis.

CXCL12/CXCR4 Signaling Enhances Human PSC-Derived Hematopoietic ProgenitorFunction and Overcomes Early InVivo Transplantation Failure.

SummaryHuman pluripotent stem cells (hPSCs) generate hematopoietic progenitor cells (HPCs) but fail to engraft xenograft models used to detect adult/somatic hematopoietic stem cells (HSCs) from donors. Recent progress to derive hPSC-derived HSCs has relied on cell-autonomous forced expression of transcription factors; however, the relationship of bone marrow to transplanted cells remains unknown. Here, we quantified a failure of hPSC-HPCs to survive even 24 hr post transplantation. Across several hPSC-HPC differentiation methodologies, we identified the lack of CXCR4 expression and function. Ectopic CXCR4 conferred CXCL12 ligand-dependent signaling of hPSC-HPCs in biochemical assays and increased migration/chemotaxis, hematopoietic progenitor capacity, and survival and proliferation following in vivo transplantation. This was accompanied by a transcriptional shift of hPSC-HPCs toward somatic/adult sources, but this approach failed to produce long-term HSC xenograft reconstitution. Our results reveal that networks involving CXCR4 should be targeted to generate putative HSCs with in vivo function from hPSCs.

Outcomes of Children with Hemophagocytic Lymphohistiocytosis Given Allogeneic Hematopoietic Stem Cell Transplantation in Italy

We report on 109 patients with hemophagocytic lymphohistiocytosis (HLH) undergoing 126 procedures of allogeneic hematopoietic stem cell transplantation (HSCT) between 2000 and 2014 in centers associated with the Italian Pediatric Hematology Oncology Association. Genetic diagnosis was FHL2 (32%), FHL3 (33%), or other defined disorders known to cause HLH (15%); in the remaining patients no genetic abnormality was found. Donor for first transplant was an HLA-matched sibling for 25 patients (23%), an unrelated donor for 73 (67%), and an HLA-partially matched family donor for 11 children (10%). Conditioning regimen was busulfan-based for 61 patients (56%), treosulfan-based for 21 (20%), and fludarabine-based for 26 children (24%). The 5-year probabilities of overall survival (OS) and event-free survival (EFS) were 71% and 60%, respectively. Twenty-six patients (24%) died due to transplant-related causes, whereas 14 (13%) and 10 (9%) patients experienced graft rejection and/or relapse, respectively. Twelve of 14 children given a second HSCT after graft failure/relapse are alive and disease-free. Use of HLA-partially matched family donors was associated with higher risk of graft failure and thus with lower EFS (but not with lower OS) in multivariable analysis. Active disease at transplantation did not significantly affect prognosis. These data confirm that HSCT can cure most HLH patients, active disease not precluding successful transplantation. Because in HLH patients HLA-haploidentical HSCT performed through CD34+ cell positive selection was found to be associated with poor sustained engraftment of donor cells, innovative approaches able to guarantee a more robust engraftment are warranted in patients given this type of allograft.

Cografting astrocytes improves cell therapeutic outcomes in a Parkinson's disease model.

Transplantation of neural progenitor cells (NPCs) is a potential therapy for treating neurodegenerative disorders, but this approach has faced many challenges and limited success, primarily because of inhospitable host brain environments that interfere with enriched neuron engraftment and function. Astrocytes play neurotrophic roles in the developing and adult brain, making them potential candidates for helping with modification of hostile brain environments. In this study, we examined whether astrocytic function could be utilized to overcome the current limitations of cell-based therapies in a murine model of Parkinson's disease (PD) that is characterized by dopamine (DA) neuron degeneration in the midbrain. We show here that cografting astrocytes, especially those derived from the midbrain, remarkably enhanced NPC-based cell therapeutic outcomes along with robust DA neuron engraftment in PD rats for at least 6 months after transplantation. We further show that engineering of donor astrocytes with Nurr1 and Foxa2, transcription factors that were recently reported to polarize harmful immunogenic glia into the neuroprotective form, further promoted the neurotrophic actions of grafted astrocytes in the cell therapeutic approach. Collectively, these findings suggest that cografting astrocytes could be a potential strategy for successful cell therapeutic outcomes in neurodegenerative disorders.

The CXCR4 Antagonist BL-8040 Induces a Robust Mobilization of CD34+CD38−CD45RA−CD90+ CD49f+ HSCs with Long-Term and Secondary Myeloid and Lymphoid Repopulating Activity

▪Introduction: Lifelong blood cell production is dependent on rare hematopoietic stem cells (HSCs) to perpetually replenish mature cells via a series of lineage-restricted intermediates.The bulk of HSCs are CD34+, however, most CD34+ cells are lineage-restricted progenitors and HSCs remain rare. HSCs can be enriched further based on CD45RA, Thy1 (CD90), and CD38 expression. Loss of CD90 expression was proposed to be sufficient to separate CD34+CD38−CD45RA− CD90+ HSCs from CD34+CD38−CD45RA− CD90- multipotent progenitors (MPPs). Recently, it was demonstrated that the expression CD49f is a specific HSC marker. Single CD49f(+) cells were highly efficient in generating long-term multilineage grafts, and the loss of CD49f expression identified transiently engrafting MPPs.Results: CD34+ cells were purified from BL-8040 and G-CSF mobilized grafts and stained for CD38, CD45RA, CD90, and CD49f. The percentage of CD34+CD38- hematopoietic stem and progenitors was similar in both grafts (Figure 1A). However, whereas 23.2 % of BL-8040 mobilized CD34+ CD38- cells did not express CD45RA; only 1.6% of G-CSF mobilized CD34+ CD38- cells did not express CD45RA (Figure 1B). The percentages of CD34+CD38−CD45RA−CD49f+CD90+/-, CD34+CD38−CD45RA−CD49f+ CD90+, and CD34+CD38−CD45RA− CD90+ HPCS were increased significantly by 45, 25 and 12 -fold in the BL-8040 graft compared to G-CSF graft derived CD34+CD38- cells (Figure 1C). To assess the long-term engraftment potential of the BL-8040 mobilized CD34+ cells, engraftment was allowed for 4, 8, and 22 weeks after transplantation. Successful and robust long-term human engraftment of CD45+ and CD45+CD34+ cells was observed at week 22 (Figure 2A, 2B). The % of human CD45 cells remained stable in the BM whereas the percentage of CD45 cells in the blood and spleen increased at week 22 (Figure 2C). At 4 weeks, human CD3+CD4+ T cells were only observed at a low percentage in the spleen but not in the BM, whereas no significant percentage of CD3+CD8+ cells were found neither in the BM nor in the spleen (Figure 2D, 2E). 22 weeks after transplantation, the percentage of human CD3+CD4+ and CD3+CD8+ T cells was significantly increased in the spleen (30% vs. 5%, respectively) and to much lower levels in the BM (Figure 2D, 2E). Furthermore, successful and robust long-term human engraftment of secondary recipient was observed 14 weeks following the second transplantation (Figure 2A and 2B).Conclusion: In association with the high percentage of HPCs in the BL-8040-derived graft, we found a robust myeloid and lymphoid long-term engraftment (week 22) of BL-8040 mobilized human CD34+ cells in NSG mice. The ability of BL-8040 to collect high numbers of HPCs may be beneficial for a variety of HPCs dependent therapeutics. [Display omitted] DisclosuresAbraham:Biokine: Employment. Oberkovitz:BiolineRx: Employment. Eizenberg:Biokine: Employment. Vainstein:BiolineRx: Employment. Benami:BiolineRx: Employment. Golan:BiolineRx: Employment. Or:Bioline: Consultancy. Peled:Biokine: Consultancy; Biosight: Consultancy.

Intracerebroventricular delivery of hematopoietic progenitors results in rapid and robust engraftment of microglia-like cells

Recent evidence indicates that hematopoietic stem and progenitor cells (HSPCs) can serve as vehicles for therapeutic molecular delivery to the brain by contributing to the turnover of resident myeloid cell populations. However, such engraftment needs to be fast and efficient to exert its therapeutic potential for diseases affecting the central nervous system. Moreover, the nature of the cells reconstituted after transplantation and whether they could comprise bona fide microglia remain to be assessed. We demonstrate that transplantation of HSPCs in the cerebral lateral ventricles provides rapid engraftment of morphologically, antigenically, and transcriptionally dependable microglia-like cells. We show that the cells comprised within the hematopoietic stem cell compartment and enriched early progenitor fractions generate this microglia-like population when injected in the brain ventricles in the absence of engraftment in the bone marrow. This delivery route has therapeutic relevance because it increases the delivery of therapeutic molecules to the brain, as shown in a humanized animal model of a prototypical lysosomal storage disease affecting the central nervous system.

Magnetic Targeting of Stem Cell Derivatives Enhances Hepatic Engraftment into Structurally Normal Liver

Attaining consistent robust engraftment in the structurally normal liver is an obstacle for cellular transplantation. Most experimental approaches to increase transplanted cells’ engraftment involve recipient-centered deleterious methods such as partial hepatectomy or irradiation which may be unsuitable in the clinic. Here, we present a cell-based strategy that increases engraftment into the structurally normal liver using a combination of magnetic targeting and proliferative endoderm progenitor (EPs) cells. Magnetic labeling has little effect on cell viability and differentiation, but in the presence of magnetic targeting, it increases the initial dwell time of transplanted EPs into the undamaged liver parenchyma. Consequently, greater cell retention in the liver is observed concomitantly with fewer transplanted cells in the lungs. These highly proliferative cells then significantly increase their biomass over time in the liver parenchyma, approaching nearly 4% of total liver cells 30 d after transplant. Therefore, the cell-based mechanisms of increased initial dwell time through magnetic targeting combined with high rate of proliferation in situ yield significant engraftment in the undamaged liver.