Successful Reconstitution Research Articles

Application of a Cell-Free Synthetic Biology Platform for the Reconstitution of Teleocidin B and UK-2A Precursor Biosynthetic Pathways.

We report the successful cell-free reconstitution of two natural product biosynthetic pathways of divergent complexity and structural classes. We first constructed the teleocidin biosynthetic pathway using our BY-2 (tobacco) cell-free protein synthesis (CFPS) system. We discovered a direct interaction between TleA and MbtH, and showed that the BY-2 system is capable of producing more than 80 mg/L teleocidin B-3 with cofactor supplementation and ∼20 mg/L with no cofactors supplemented, demonstrating the high metabolic activity of the system. We then extended our methodology and report the first successful cell-free biosynthesis of UK-2 diol (precursor to the commercially valuable secondary metabolite UK-2A) from simple building blocks by refactoring a complex pathway of 10 proteins in the wheat germ CFPS system. We show that plant CFPS systems are suitable for reconstructing pathways and identifying the functions of uncharacterized genes linked to biosynthetic gene clusters and rate-limiting biosynthetic steps.

A synthetic cytotoxic T cell platform for rapidly prototyping TCR function

Current tools for functionally profiling T cell receptors with respect to cytotoxic potency and cross-reactivity are hampered by difficulties in establishing model systems to test these proteins in the contexts of different HLA alleles and against broad arrays of potential antigens. We have implemented a granzyme-activatable sensor of T cell cytotoxicity in a universal prototyping platform which enables facile recombinant expression of any combination of TCR-, peptide-, and class I MHC-coding sequences and direct assessment of resultant responses. This system consists of an engineered cell platform based on the immortalized natural killer cell line, YT-Indy, and the MHC-null antigen-presenting cell line, K562. These cells were engineered to furnish the YT-Indy/K562 pair with appropriate protein domains required for recombinant TCR expression and function in a non-T cell chassis, integrate a fluorescence-based target-centric early detection reporter of cytotoxic function, and deploy a set of protective genetic interventions designed to preserve antigen-presenting cells for subsequent capture and downstream characterization. Our data show successful reconstitution of the surface TCR complex in the YT-Indy cell line at biologically relevant levels. We also demonstrate successful induction and highly sensitive detection of antigen-specific response in multiple distinct model TCRs. Additionally, we monitored destruction of targets in co-culture and found that our survival-optimized system allowed for complete preservation after 24 h exposure to cytotoxic effectors. With this bioplatform, we anticipate investigators will be empowered to rapidly express and characterize T cell receptor responses, generate knowledge regarding the patterns of T cell receptor recognition, and optimize therapeutic T cell receptors.

Isolation of Human Hematopoietic Stem Cells from an Apheresis Sample.

The hematopoietic system constantly produces new blood cells through hematopoiesis, and maintaining this balance is vital for human health. This balance is maintained by self-renewing hematopoietic stem cells (HSCs) and various progenitor cells. Under typical circumstances, HSCs are not abundantly found in peripheral blood; hence, their mobilization from the bone marrow is vital. Hematopoietic growth factors achieve this effectively, enabling mobilization and thus allowing blood sample and thus HSC collection via apheresis. Securing a sufficient supply of HSCs is vital for successful hematopoietic reconstitution and the rapid integration of committed cells. Thus, isolation and expansion of HSCs are crucial for convenient extraction, production of transplantable quantities, genetic modifications for enhanced therapeutic efficacy, and as a source of increased/expanded/synthesized blood cells in vitro. In conclusion, the isolation and expansion of HSCs play pivotal roles in both regenerative medicine and hematology. This protocol describes the isolation of human HSCs by providing an overview of the primary method for isolating human hematopoietic stem cells from apheresis blood samples and sheds light on human HSC studies and developments in research and medicine.

Successful Immune Reconstitution in a Patient with a TYK2 Deficiency after Allogeneic Stem Cell Transplantation from Unrelated Donors.

A boy with primary immunodeficiency, caused by a tyrosine kinase 2 (TYK2) mutation, presented with immune defects and a lifelong history of severe infections. Our aim was to determine whether allogeneic hematopoietic stem cell transplantation (HSCT) could restore the patient's immune defenses and reduce susceptibility to infection. In the absence of a suitable HLA-matched blood relative to act as a donor, the patient received an allogeneic HSCT from unrelated donors. The patient's clinical data were analyzed in the Children's Hospital of Chongqing Medical University (Chongqing, China) before transplantation and during the 4-year follow-up period using a combination of western blotting (e.g., TYK2 and STAT levels), qRT-PCR (e.g., T cell receptor rearrangement excision circles, kappa deletion element recombination circles, and TYK2 transcript levels), and flow cytometry (e.g., lymphocyte subpopulations and CD107α secretion). We found that HSCT significantly reduced the incidence of severe infections, restored normal TKY2 levels, and reversed defects such as impaired JAK/STAT signaling in response to interferon-α or interleukin-10 treatment. Although the patient did not develop acute graft-versus-host disease (GVHD) after transplantation, he did experience chronic GVHD symptoms in a number of organs, which were effectively managed. Our findings suggest that HSCT is a feasible strategy for reconstituting the immune system in TYK2-deficient patients; however, the factors associated with GVHD and autoimmune thyroiditis development in TYK2-deficient patients undergoing HSCT warrant further investigation.

Evolution of paralogous multicomponent systems for site-specific O-sialylation of flagellin in Gram-negative and Gram-positive bacteria

Many bacteria glycosylate flagellin on serine or threonine residues using pseudaminic acid (Pse) or other sialic acid-like donor sugars. Successful reconstitution of Pse-dependent sialylation by the conserved Maf-type flagellin glycosyltransferase (fGT) may require (a)missing component(s). Here, we characterize both Maf paralogs in the Gram-negative bacterium Shewanella oneidensis MR-1 and reconstitute Pse-dependent glycosylation in heterologous hosts. Remarkably, we uncovered distinct acceptor determinants and target specificities for each Maf. Whereas Maf-1 uses its C-terminal tetratricopeptide repeat (TPR) domain to confer flagellin acceptor and O-glycosylation specificity, Maf-2 requires the newly identified conserved specificity factor, glycosylation factor for Maf (GlfM), to form a ternary complex with flagellin. GlfM orthologs are co-encoded with Maf-2 in Gram-negative and Gram-positive bacteria and require an invariant aspartate in their four-helix bundle to function with Maf-2. Thus, convergent fGT evolution underlies distinct flagellin-binding modes in tripartite versus bipartite systems and, consequently, distinct O-glycosylation preferences of acceptor serine residues with Pse.

Engineered Biosynthesis and Anticancer Studies of Ring-Expanded Antimycin-Type Depsipeptides.

Respirantins are 18-membered antimycin-type depsipeptides produced by Streptomyces sp. and Kitasatospora sp. These compounds have shown extraordinary anticancer activities against a panel of cancer cell lines with nanomolar levels of IC50 values. However, further investigation has been impeded by the low titers of the natural producers and the challenging chemical synthesis due to their structural complexity. The biosynthetic gene cluster (BGC) of respirantin was previously proposed based on a bioinformatic comparison of the four members of antimycin-type depsipeptides. In this study, we report the first successful reconstitution of respirantin in Streptomyces albus using a synthetic BGC. This heterologous system serves as an accessible platform for the production and diversification of respirantins. Through polyketide synthase pathway engineering, biocatalysis, and chemical derivatization, we generated nine respirantin compounds, including six new derivatives. Cytotoxicity screening against human MCF-7 and Hela cancer cell lines revealed a unique biphasic dose-response profile of respirantin. Furthermore, a structure-activity relationship study has elucidated the essential functional groups that contribute to its remarkable cytotoxicity. This work paves the way for respirantin-based anticancer drug discovery and development.

A new preparation method of covalent annular nanodiscs based on MTGase

The preservation of the native conformation and functionality of membrane proteins has posed considerable challenges. While detergents and liposome reconstitution have been traditional approaches, nanodiscs (NDs) offer a promising solution by embedding membrane proteins in phospholipids encircled by an amphipathic helical protein MSP belt. Nevertheless, a drawback of commonly used NDs is their limited homogeneity and stability. In this study, we present a novel approach to construct covalent annular nanodiscs (cNDs) by leveraging microbial transglutaminase (MTGase) to catalyze isopeptide bond formation between the side chains of terminal amino acids, specifically Lysine (K) and Glutamine (Q). This methodology significantly enhances the homogeneity and stability of NDs. Characterization of cNDs and the assembly of membrane proteins within them validate the successful reconstitution of membrane proteins with improved homogeneity and stability. Our findings suggest that cNDs represent a more suitable tool for investigating interactions between membrane proteins and lipids, as well as for analyzing membrane protein structures.

Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System.

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting the N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful cotranslational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells.

Recombinant Human Thrombopoietin Promotes Platelet Engraftment in Severe Aplastic Anemia Patients Following Treatment With Haploid Hematopoietic Stem Cell Transplantation using Modified Post-Transplantation Cyclophosphamide

BackgroundRecombinant human TPO (rhTPO) promotes platelet engraftment in patients after allogeneic HSCT (allo-HSCT). However, the effects of rhTPO on platelet recovery after Haplo-HSCT in patients with severe aplastic anemia (SAA) have not been intensively studied. ObjectiveWe aimed to evaluate the efficacy of rhTPO on platelet engraftment in patients with SAA who were treated with Haplo-HSCT using post-transplantation cyclophosphamide (PTCy). Study DesignSAA patients who received Haplo-HSCT plus PTCy regimen were divided into the rhTPO group (with subcutaneous injection of rhTPO, n = 28) and Control group (no rhTPO administration, n = 27). The engraftment of platelet/neutrophil, platelet infusion amount, and transplant-related complications between the 2 groups were compared. ResultsAll 55 patients showed successful hematopoietic reconstitution. The median time of platelet engraftment was 11 (9 to 29) days in the rhTPO group and 14 (9 to 28) days in the Control group (P = .003). The rhTPO group had a significantly reduced amount of infused platelets compared to the Control group (2 (1 to 11.5) versus 3 (1 to 14) therapeutic doses; P = .004). There was no significant difference between the 2 groups regarding median time of neutrophil engraftment, incidence of acute graft-versus-host disease (aGVHD) and chronic GVHD (cGVHD), incidence of cytomegalovirus or Epstein–Barr virus reactivation, 3-yr overall survival rate, and failure-free-survival rate. No obvious adverse reactions were observed in the rhTPO group. ConclusionrhTPO promoted platelet engraftment, reduced the amount of transfused platelets, and demonstrated good safety profiles without evidence of adverse reactions in patients with SAA who received Haplo-HSCT using PTCy regimen.

Ulnar Nerve Reconstruction With a Basilic Vein Tributary Graft: A Practical and Easily Replicable Technique With Favourable Outcomes

A young gentleman presented with a closed fracture of the left radius and an open fracture of the left ulna complicated with segmental loss of the ulnar nerve measuring 1.5cm. After thorough debridement and stabilization of the bone injuries, the ulnar nerve gap was bridged with an autologous venous graft harvested off a tributary of the basilic vein that served as a nerve conduit. At 18 months post-surgery, sensori-motor function of the patient’s left ulnar nerve was nearly fully restored, indicative of successful reconstitution of the ulnar nerve using a basilic vein tributary vein graft.

An engineered thermally tolerant apo-cytochrome scaffold for metal-less incorporation of heme derivative.

Cytochrome c552 from Thermus thermophilus is one of the hot topics for creating smart biomaterials as it possesses remarkable stability, is tolerant to multiple mutations and has therefore been recently reported for a number of functionalizations upon substitution of the original prosthetic group with an artificial prosthetic group. However, all of the substitutions were driven by the coordination through the axial ligands followed by complete reconstitution with a metal-porphyrin complex. This limits the scope of the cytochrome c for incorporating a metal-less non-natural heme species that could improve the versatility of cytochrome c for a new generation of engineered cytochrome proteins for further enhancement in their functionalities such as biocatalysts. In this connection, a new variant of Cytochrome c (rC552 C14A) from Thermus thermophilus was reported, where an easy approach to remove the original prosthetic group was achieved, followed by the incorporation of a number of metal-PPIX derivatives that ultimately led to the formation of artificial c-type cytochromes through covalent bonding. The apo-cytochrome was found to be thermally tolerant and to possess a distinctive overall structure as that of the wild type, as was evident from the corresponding CD spectra, which ultimately encouraged reconstitution with a metal-less protoporphyrin derivative for better understanding the role of axial ligands in the reconstitution process. Successful reconstitution was achieved, resulting in a new type of Cytochrome b-type artificial protein without the metal in its active site, indicating the non-involvement of the axial ligand. In order to prove the non-involvement of the axial ligand, a subsequent double mutant (C14A/M69A) was constructed, replacing the methionine at 69 position with non-coordinating alanine residue. Accordingly, the apo-C14A/M69A was prepared and found to be extremely stable as the earlier mutants and the WT showed no signs of denaturation, even at the elevated temperature of 98°C. Subsequently, heme b was successfully incorporated into the apo-C14A/M69A, which demonstrated itself as a highly thermally tolerant protein scaffold for incorporating a metal-less artificial prosthetic group in the absence of the axial ligand. Further improvement in the reconstitution process is achieved by replacing the methionine at 69 position with phenyl alanine (C14A/M69F mutant), resulting in further stabilization of heme species, possibly through non-covalent π-interactions, as corroborated by molecular docking.

Reconstitution of actin-based cellular processes: Why encapsulation changes the rules

While in vitro reconstitution of cellular processes is progressing rapidly, the encapsulation of biomimetic systems to reproduce the cellular environment is a major challenge. Here we review the difficulties, using reconstitution of processes dependent on actin polymerization as an example. Some of the problems are purely technical, due to the need for engineering strategies to encapsulate concentrated solutions in micrometer-sized compartments. However, other significant issues arise from the reduction of experimental volumes, which alters the chemical evolution of these non-equilibrium systems. Important parameters to consider for successful reconstitutions are the amount of each component, their consumption and renewal rates to guarantee their continuous availability.

Cefiderocol For Injection: Compatibility Testing Using the MINI-BAG Plus Container System and the VIAL-MATE Adaptor.

Background: Cost-effective and convenient modalities are required to facilitate the administration of antibiotics in hospital and outpatient settings. Objective: This study investigated the physical compatibility of the MINI-BAG Plus Container System and VIAL-MATE Adaptor with the 1 g drug product vials used for cefiderocol. Methods: Qualitative testing of the MINI-BAG Plus Container System (50 and 100 mL of 5% dextrose injection or 0.9% sodium chloride injection), using empty vials and vials containing lyophilized cefiderocol powder, was conducted in triplicate on MINI-BAGs that were hung and observed over 3 hours. Connection security between empty vials and the VIAL-MATE Adaptor was assessed in triplicate. Results: All predefined physical compatibility criteria between cefiderocol 1 g vials and the MINI-BAG Plus Container System were met, including a secure connection, successful multiple transfers of solution between vial and bag, successful reconstitution of cefiderocol, and lack of leaking into the vial or from the connections. There was no particulate matter in the prepared solution and no precipitation or discoloration. Secure connections between the VIAL-MATE Adaptor and cefiderocol vials were demonstrated. Conclusion and Relevance: Use of these systems is relevant even where resources are limited and may increase the efficiency of cefiderocol administration in hospitals, outpatient settings, or long-term healthcare facilities.

Base-Edited CAR7 T Cells for Relapsed T-Cell Acute Lymphoblastic Leukemia

BackgroundCytidine deamination that is guided by clustered regularly interspaced short palindromic repeats (CRISPR) can mediate a highly precise conversion of one nucleotide into another — specifically, cytosine to thymine — without generating breaks in DNA. Thus, genes can be base-edited and rendered inactive without inducing translocations and other chromosomal aberrations. The use of this technique in patients with relapsed childhood T-cell leukemia is being investigated.MethodsWe used base editing to generate universal, off-the-shelf chimeric antigen receptor (CAR) T cells. Healthy volunteer donor T cells were transduced with the use of a lentivirus to express a CAR with specificity for CD7 (CAR7), a protein that is expressed in T-cell acute lymphoblastic leukemia (ALL). We then used base editing to inactivate three genes encoding CD52 and CD7 receptors and the β chain of the αβ T-cell receptor to evade lymphodepleting serotherapy, CAR7 T-cell fratricide, and graft-versus-host disease, respectively. We investigated the safety of these edited cells in three children with relapsed leukemia.ResultsThe first patient, a 13-year-old girl who had relapsed T-cell ALL after allogeneic stem-cell transplantation, had molecular remission within 28 days after infusion of a single dose of base-edited CAR7 (BE-CAR7). She then received a reduced-intensity (nonmyeloablative) allogeneic stem-cell transplant from her original donor, with successful immunologic reconstitution and ongoing leukemic remission. BE-CAR7 cells from the same bank showed potent activity in two other patients, and although fatal fungal complications developed in one patient, the other patient underwent allogeneic stem-cell transplantation while in remission. Serious adverse events included cytokine release syndrome, multilineage cytopenia, and opportunistic infections.ConclusionsThe interim results of this phase 1 study support further investigation of base-edited T cells for patients with relapsed leukemia and indicate the anticipated risks of immunotherapy-related complications. (Funded by the Medical Research Council and others; ISRCTN number, ISRCTN15323014.)

Successful treatment of DOCK8 deficiency by allogeneic hematopoietic cell transplantation from alternative donors.

Dedicator of cytokinesis 8 (DOCK8) deficiency is a rare autosomal recessive inborn error of immunity (IEI) characterized by eczematous dermatitis, elevated serum IgE, and recurrent infections, comprising a seemingly hyper-IgE syndrome (HIES). DOCK8 deficiency is only curable with allogeneic hematopoietic cell transplantation (HCT), but the outcome of HCT from alternative donors is not fully understood. Here, we describe the cases of two Japanese patients with DOCK8 deficiency who were successfully treated by allogeneic HCT from alternative donors. Patient 1 underwent cord blood transplantation at the age of 16years, and Patient 2 underwent haploidentical peripheral blood stem cell transplantation with post-transplant cyclophosphamide at the age of 22years. Each patient received a fludarabine-based conditioning regimen. Their clinical manifestations, including refractory molluscum contagiosum, promptly improved post-HCT. They achieved successful engraftment and immune reconstitution without serious complications. Alternative donor sources such as cord blood and haploidentical donors can be options for allogeneic HCT for DOCK8 deficiency.

Chromosome 22q11.2 Deletion (DiGeorge Syndrome): Immunologic Features, Diagnosis, and Management.

This review focuses on immunologic findings, relationships among immunologic findings and associated conditions of autoimmunity and atopy, and management of immunologic disease in chromosome 22q11.2 deletion syndrome (22q11.2DS, historically known as DiGeorge syndrome). The implementation of assessment of T cell receptor excision circles (TRECs) in newborn screening has led to increased detection of 22q11.2 deletion syndrome. While not yet applied in clinical practice, cell-free DNA screening for 22q11.2DS also has the potential to improve early detection, which may benefit prompt evaluation and management. Multiple studies have further elucidated phenotypic features and potential biomarkers associated with immunologic outcomes, including the development of autoimmune disease and atopy. The clinical presentation of 22q11.2DS is highly variable particularly with respect to immunologic manifestations. Time to recovery of immune system abnormalities is not well-defined in current literature. An understanding of the underlying causes of immunologic changes found in 22q11.2DS, and the progression and evolution of immunologic changes over the lifespan have expanded over time and with improved survival. An included case highlights the variability of presentation and potential severity of T cell lymphopenia in partial DiGeorge syndrome and demonstrates successful spontaneous immune reconstitution in partial DiGeorge syndrome despite initial severe T cell lymphopenia.

Reconstitution of depolarization-induced Ca2+ release for validation of skeletal muscle disease mutations and drug discovery.

In skeletal muscle, depolarization of the plasma membrane triggers Ca2+ release from the sarcoplasmic reticulum (SR),referred to as depolarization-induced Ca2+ release (DICR). DICR occurs via the type 1 ryanodine receptor (RyR1), which physically interacts with the dihydropyridine receptor Cav1.1 subunit in specific machinery formed with additional essential components including β1a, Stac3 adaptor protein and junctophilins. It has recently become clear that mutations in these components cause various skeletal muscle diseases. However, functional validation is time-consuming because it must be performed in a skeletal muscle environment and no specific treatment has been developed yet. In this study, we established a platform of the reconstituted DICR in HEK293 cells. The essential components were effectively transduced using baculovirus vectors, and Ca2+ release was quantitatively measured with R-CEPIA1er, a fluorescent ER Ca2+indicator. High [K+] depolarization triggered rapid Ca2+ release, indicating successful reconstitution of DICR. We evaluated several Cav1.1 mutations that are implicated in malignant hyperthermia and myopathy. We also tested several RyR1 inhibitors; whereas dantrolene and Cpd1 inhibited DICR, procaine had no effect. These results well reproduced the findings with the muscle fibers and the cultured myotubes. Since the procedure is simple and reproducible, the reconstituted DICR platform will be highly useful for validation of mutations and drug discovery for skeletal muscle diseases.

Stiffness-Controlled Hydrogels for 3D Cell Culture Models.

Nanofibrillated cellulose (NFC) hydrogel is a versatile biomaterial suitable, for example, for three-dimensional (3D) cell spheroid culturing, drug delivery, and wound treatment. By freeze-drying NFC hydrogel, highly porous NFC structures can be manufactured. We freeze-dried NFC hydrogel and subsequently reconstituted the samples into a variety of concentrations of NFC fibers, which resulted in different stiffness of the material, i.e., different mechanical cues. After the successful freeze-drying and reconstitution, we showed that freeze-dried NFC hydrogel can be used for one-step 3D cell spheroid culturing of primary mesenchymal stem/stromal cells, prostate cancer cells (PC3), and hepatocellular carcinoma cells (HepG2). No difference was observed in the viability or morphology between the 3D cell spheroids cultured in the freeze-dried and reconstituted NFC hydrogel and fresh NFC hydrogel. Furthermore, the 3D cultured spheroids showed stable metabolic activity and nearly 100% viability. Finally, we applied a convolutional neural network (CNN)-based automatic nuclei segmentation approach to automatically segment individual cells of 3D cultured PC3 and HepG2 spheroids. These results provide an application to culture 3D cell spheroids more readily with the NFC hydrogel and a step towards automatization of 3D cell culturing and analysis.

Preliminary Safety and Efficacy Results of EDI001: An Investigator Initiated Trial on CRISPR/Cas9-Modified Autologous CD34+ Hematopoietic Stem and Progenitor Cells for Patients with Transfusion Dependent β-Thalassemia

Background In China, it is estimated that there are 15,000-30,000 patients with β Thalassemia Major (β-TM). Our recent Cross-sectional survey of β-TM patients’ conditions in China found that only 7% of patients with β-TM expect to live beyond age 18, and only a fraction of patients have received allogeneic hematopoietic stem cell transplantation. Higher level of fetal hemoglobin (HbF) by downregulation of BCL11A during erythroid differentiation has been reported to ameliorate the clinical severity of the patients with β-TM. Disruption of GATAA motif in DHS h+58 region of BCL11A erythroid enhancer (BEE) by gene editing in CD34+ cells has been shown to significantly induce the production of HbF in patients. An adjacent motif, i.e., the CTG motif within TAL1-GATA1 binding site could potentially also be such a gene-editing target due to naturally occurring SNPs associated with elevated HbF levels (eg rs1427407). ET-01 is an investigational cell therapy product comprised of autologous CD34+ cells that have undergone CRISPR/Cas9-mediated ex vivo editing targeting this motif (Fig 1). Methods The EDI-001 trial (NCT04390971) aimed to evaluate the safety and efficacy of ET-01 in patients with transfusion dependent thalassemia (TDT). Patients (aged 6 to 35 years) with TDT whose total hemoglobin level fell within the range of 4.5g/dL and 7g/dL were eligible. One patient was enrolled and CD34+ cells were collected from the patient by apheresis following mobilization with lenograstim and plerixafor. ET-01 was manufactured by editing at BEE with Cas9 mRNA and a specific single-guide RNA via electroporation. The ET-01 product was infused following myeloablative conditioning on date 18 Nov 2020 (patient age at 12). Afterwards, the patient was followed up per schedule defined in the Protocol, monitoring safety and efficacy endpoints. Results cGMP manufacturing process at clinical scale was developed for ET-01. High editing rate was achieved with stemness well maintained (Fig 2A). Indels pattern analysis showed that in ET-01 four indel types were most prevalent (Fig 2B). Moreover, the percentage of HbF+ cells were significantly increased compared with the unedited CD34+ cells in erythroid differentiation assay (Fig 2C). IND-enabling studies showed that no tumorigenicity nor other toxicities associated with ET-01 were observed in over 400 mice at up to 40 weeks of observation. One patient (β0/β+) was enrolled, for which ET-01 was manufactured under cGMP process and infused at a dose of 23.19 X 10^6 CD34+ cells/kg. Successful hematopoietic reconstitution was observed, in which neutrophil engraftment occurred on day 24 and platelet engraftment on day 37 post ET-01 infusion. The patient experienced a significant increase of HbF production and total Hb, in which the levels of HbF increased from 3.35 g/L at baseline to 89.87 g/L, and the total hemoglobin level reached 110 g/L at month 18, respectively (Fig 3). The patient received her last pRBCs transfusion on day 87 after ET-01 infusion, and achieved transfusion-free for over 15 months. Moreover, we observed that the prevalence of indels changed during the initial few weeks and remained stable in both peripheral blood and bone marrow (Fig 4A). NGS analysis of 42 potential off-target sites, discovered by in silico prediction and DiGenome-seq, showed editing efficiencies below 1%, and no significant change was observed in any of the 42 sites throughout visits (Fig 4B). The patient experienced 62 cases of adverse events, most of which were Grade 1 or 2 in severity. Of these, only one Grade 1 AE was attributed to the ET-01 drug product, caused by the ET-01 preparation component DMSO during infusion, and was gradually relieved by adjusting the infusion rate. Two Grade 3 serious adverse events (SAE) unrelated to ET-01 were reported: enterocolitis and febrile neutropenia, caused by inappropriate diet after apheresis and busulfan conditioning, respectively. Conclusions These data demonstrated that a single-dose of ET-01 composed of autologous CRISPR/Cas9 mediated CD34+ cells led to the timely hematopoietic engraftment, significant and durable increases in HbF production and total Hb, and transfusion-independent for 15 months at the time of data cut-off. Safety profile of ET-01 is consistent with that of autologous HSC transplant including busulfan myeloablative conditioning. These preliminary results support further experimental testing of ET-01 to treat patients with β-TM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Distinct Patterns in the Reconstitutions of Treg and T-Helper Subsets after Autologous Hematopoietic Stem Cell Transplantation in Juvenile-Onset Systemic Sclerosis Patients

Systemic sclerosis (SSc) is a refractory autoimmune disease characterized by diffuse fibrosis and vasculopathy affecting the skin, joints, and internal organs. Most immunosuppressive drugs have failed to show long-term benefit in randomized trials. In recent years, autologous hematopoietic stem cell transplantation (ASCT) has been performed to treat multiple sclerosis and SSc, however, the reconstitutions of immune components post-ASCT in SSc have not been fully described. Subjects and Methods: We tested a total of 13 samples from 4 consenting SSc patients (3 females, 1 male) receiving IRB-approved immunoablation (TBI 600cGy) with lung shielding and Cytoxan (n=3) or Thiotepa (n=1) +/- Alemtuzumab followed by autologous CD34+ selected, cryopreserved peripheral blood hematopoietic stem cells transplantation. Their median age was 18.5-years-old (range 15.2- to 21.4-years-old). Peripheral blood samples were collected at baseline pre-ASCT, and 3, 6, 12, 24-months post-ASCT. By flow cytometry, we examined lymphocyte and monocyte subset recovery with additional focus on the FOXP3+Treg and T-helper subset reconstitution. Results and Discussion: All 4 subjects experienced significant improvement in several clinical and quality of life parameters, especially in their skin disease evident within weeks after ASCT. T-cell lymphopenia was profound at 3-months; however, the recovery was evident by 6-months reaching pre-ASCT levels by 2-years post-ASCT (Fig 1A). Natural killer (NK) lymphocytes and monocytes attained the normal values by 3-months and after (Fig 1A). The FOXP3+Tregs that appeared post-ASCT displayed distinct features. There was an increased percentage of FOXP3+Tregs in total CD4+T cells during the early stage (3- to 6-months) post-ASCT (Fig 1B), leading to an increase in the ratio of Treg/Tcon (Fig 1B). The higher proportions of proliferating Tregs as measured by Ki67+FOXP3+CD4+/CD25+/CD127- T cells (Fig 1C) likely led to their increased numbers. This trend was sustained until the last evaluation at 2-years post-ASCT (Fig 1C). In contrast, CD4+Tcons showed a relatively lower proliferation rate, leading to a higher ratio of Ki67+Treg/Ki67+Tcon compared to pre-ASCT values (Fig 1C). Notably, there was an increased proportion of Tregs expressing the skin homing marker cutaneous lymphocyte-associated antigen (CLA) at 3-months lasting until the last test at 2-years post-ASCT (Fig 1D), whereas CLA+/CD4+Tcons were less plentiful. The reconstitutions of T-helper (Th) subsets were also examined. Due to lymphopenia at the early stage post-ASCT, Th subsets were identified according to their chemokine receptor expression (Mahnke et alCytometry 2013;83(5):439-440). Gradually decreased numbers of Th2, Th9, and Th17 were observed in the peripheral pool (Fig 1E) contributing to a decline of their proportions in total CD4+T (Fig 1F). Nevertheless, absolute number of Th1 and Th22 cells returned to levels seen pre-ASCT without any recurrence or flare of SSc symptoms. The CD45RO+ memory phenotype was dominant during the early stages post-ASCT in all subsets (data not shown). Summary: Successful reconstitution of lymphocytes and monocytes post-ASCT was observed in available SSc patients. Post-ASCT circulating FOXP3+Tregs displayed distinct features where the increased number led to an elevated Treg/Tcon ratio in the peripheral pool. Amongst Tregs, the skin homing CLA+ subset was further enriched correlating with improved skin scores in all patients. Improved peripheral proliferation and increased expression of CLA+ Tregs may provide protection from skin disease recurrence despite parallel Tcon reconstitution. The decrease of the Th2/Th9/Th17 subsets in the peripheral pool post-ASCT possibly contributes to the clinically less autoimmune phenotype. These results need to be validated in future patients. Acknowledgements: We thank all participating patients, nurses, physicians, and scientists in this study for their contributions. The study was supported by the "Idea Development Award" from the Department of Defense - Scleroderma Research Program (Award number W81XWH-21-1-0782 and W81XWH-21-1-0783). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal