Genetic ablation of adhesion ligands mitigates rejection of allogeneic cellular immunotherapies.

Allogeneic cellular immunotherapies hold promise for broad clinical implementation but face limitations due to potential rejection of donor cells by the host immune system. Silencing of beta-2 microglobulin (B2M) expression is commonly employed to evade T cell-mediated rejection by the host, although the absence of B2M is expected to trigger missing-self responses by host natural killer (NK) cells. Here, we demonstrate that genetic deletion of the adhesion ligands CD54 and CD58 in B2M-deficient chimeric antigen receptor (CAR) T cells and multi-edited induced pluripotent stem cell (iPSC)-derived CAR NK cells reduces their susceptibility to rejection by host NK cells in vitro and in vivo. The absence of adhesion ligands limits rejection in a unidirectional manner in B2M-deficient and B2M-sufficient settings without affecting the antitumor functionality of the engineered donor cells. Thus, these data suggest that genetic ablation of adhesion ligands effectively alleviates rejection by host immune cells, facilitating the implementation of universal immunotherapy.

Clearance of residual genome editing components used for ex vivo genome-editing of allogeneic cell therapy products.

BACKGROUND AIMS: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated genome editing (GE) components (e.g., nucleases, guide RNAs (gRNAs), and plasmids) are used to genetically modify cells during development of ex vivo genome-edited cell therapies. Prolonged presence of GE components may increase the risk of unintended genome modifications (e.g., off-target editing and chromosomal rearrangements). This risk is a function of the stability of the GE components, culture conditions (i.e., culture length, media changes, etc.), and the nature of the GE component (i.e., only plasmids can be integrated into a cell's genome). Testing for residual GE components on ex vivo genetically edited drug products is generally recommended in current regulatory guidance (CBER 2024). For allogenic cell therapies derived from induced pluripotent stem cells (iPSC), cells typically undergo clonal selection and extensive culturing following completion of genome editing. This post-engineering clonal selection substantially reduces the amount of residual GE components while the long-duration cell culture significantly reduces the presence of active residual GE components. Here we present a case in which the need for testing of the drug product for residual GE components has been eliminated. METHODS: In silico modeling was used to estimate clearance mechanisms across a variety of relevant assumptions, including disposition of extracellular GE components via media changes and dilution of intracellular GE components via cell expansion. Determining the ability of each GE component-alone or in complex with other GE components-to modify genomic material was assessed by a series of both in vitro and ex vivo (i.e., engineering cells) studies. For the in vitro studies, a DNA cutting assay was developed to assess the ability of the component to cut a representative DNA strand. For the ex vivo modification of cells, an assessment of the knock-out of the relevant gene was completed by flow cytometry specifically assessing the presence or absence of protein expression on the modified cells. The persistence and stability of GE components were examined under cell-mimicking conditions and in ex vivo modified cells. The components were stressed under multiple conditions mimicking a range of culture conditions and tested in the aforementioned DNA cutting assay. The presence of residual gRNA was directly assessed in the ex vivo modified cells via a gRNA-specific digital droplet polymerase chain reaction (ddPCR) assay. RESULTS: Simulations estimating genome editing residual clearance via dilution for extracellular residuals (via media changes) or intracellular residuals (via cell doubling) demonstrate clearance of measurable residuals within 28 days of cell culture. Studies simulating the stability of genome editing residuals estimate less than 7 days for the nuclease, gRNA and ribonucleoprotein (RNP) complex. gRNA was undetectable by 8 days post-engineering under actual engineering conditions. Additionally, without gRNA present, CRISPR Cas12a nucleases did not demonstrate evidence of cutting. In contrast, plasmid DNA can be randomly integrated into the genome and free plasmid is highly stable under cell culture-like conditions (50+ days). Additionally, plasmid DNA integrated in cells will propagate during mitosis, leading to the additional risk of expansion of an unintentional integration event. CONCLUSIONS: Both the gRNA and nuclease in the RNP complex are required for DNA cutting. Neither individual component nor the complex are stable beyond 7 days in culture-mimicking conditions. These findings suggest that the risk of unplanned genomic modification resulting from residual gRNA or nuclease is minimal for processes in which extensive culture is performed after the completion of genome editing and clonal selection. However, the risk of residual plasmid DNA integration is significantly higher regardless of the manufacturing process. The residual plasmid itself is quite stable (at least 50 days) and the risk of random, off-target integration is present. By establishing the stability of these components, we have demonstrated that testing for residual gRNA or nuclease is not warranted for clonally derived allogeneic cell therapies.

An aptamer-mediated base editing platform for simultaneous knockin and multiple gene knockout for allogeneic CAR-T cells generation.

Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. In conventional gene editing platforms that rely on nuclease activity, such as clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply a novel modular RNA aptamer-mediated Pin-point base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells. We demonstrate high editing efficiency and purity at all target sites and significantly reduced frequency of chromosomal translocations compared with the conventional CRISPR-Cas9 system. Site-specific knockin of a chimeric antigen receptor and multiplex gene knockout are achieved within a single intervention and without the requirement for additional sequence-targeting components. The ability to perform complex genome editing efficiently and precisely highlights the potential of the Pin-point platform for application in a range of advanced cell therapies.

Engineering allorejection-resistant CAR-NKT cells from hematopoietic stem cells for off-the-shelf cancer immunotherapy.

The clinical potential of current FDA-approved chimeric antigen receptor (CAR)-engineered T (CAR-T) cell therapy is encumbered by its autologous nature, which presents notable challenges related to manufacturing complexities, heightened costs, and limitations in patient selection. Therefore, there is a growing demand for off-the-shelf universal cell therapies. In this study, we have generated universal CAR-engineered NKT (UCAR-NKT) cells by integrating iNKT TCR engineering and HLA gene editing on hematopoietic stem cells (HSCs), along with an ex vivo, feeder-free HSC differentiation culture. The UCAR-NKT cells are produced with high yield, purity, and robustness, and they display a stable HLA-ablated phenotype that enables resistance to host cell-mediated allorejection. These UCAR-NKT cells exhibit potent antitumor efficacy to blood cancers and solid tumors, both in vitro and in vivo, employing a multifaceted array of tumor-targeting mechanisms. These cells are further capable of altering the tumor microenvironment by selectively depleting immunosuppressive tumor-associated macrophages and myeloid-derived suppressor cells. In addition, UCAR-NKT cells demonstrate a favorable safety profile with low risks of graft-versus-host disease and cytokine release syndrome. Collectively, these preclinical studies underscore the feasibility and significant therapeutic potential of UCAR-NKT cell products and lay a foundation for their translational and clinical development.

Gene-edited cells: novel allogeneic gene/cell therapy for epidermolysis bullosa.

Epidermolysis bullosa (EB) is a group of rare genetic skin fragility disorders, which are hereditary. These disorders are associated with mutations in at least 16 genes that encode components of the epidermal adhesion complex. Currently, there are no effective treatments for this disorder. All current treatment approaches focus on topical treatments to prevent complications and infections. In recent years, significant progress has been achieved in the treatment of the severe genetic skin blistering condition known as EB through preclinical and clinical advancements. Promising developments have emerged in the areas of protein and cell therapies, such as allogeneic stem cell transplantation; in addition, RNA-based therapies and gene therapy approaches have also become a reality. Stem cells obtained from embryonic or adult tissues, including the skin, are undifferentiated cells with the ability to generate, maintain, and replace fully developed cells and tissues. Recent advancements in preclinical and clinical research have significantly enhanced stem cell therapy, presenting a promising treatment option for various diseases that are not effectively addressed by current medical treatments. Different types of stem cells such as primarily hematopoietic and mesenchymal, obtained from the patient or from a donor, have been utilized to treat severe forms of diseases, each with some beneficial effects. In addition, extensive research has shown that gene transfer methods targeting allogeneic and autologous epidermal stem cells to replace or correct the defective gene are promising. These methods can regenerate and restore the adhesion of primary keratinocytes in EB patients. The long-term treatment of skin lesions in a small number of patients has shown promising results through the transplantation of skin grafts produced from gene-corrected autologous epidermal stem cells. This article attempts to summarize the current situation, potential development prospects, and some of the challenges related to the cell therapy approach for EB treatment.

The Upscale Manufacture of Chondrocytes for Allogeneic Cartilage Therapies.

Allogeneic chondrocyte therapies need to be developed to allow more individuals to be treated with a cell therapy for cartilage repair and to reduce the burden and cost of the current two-stage autologous procedures. Upscale manufacture of chondrocytes using a bioreactor could help provide an off-the-shelf allogeneic chondrocyte therapy with many doses being produced in a single manufacturing run. In this study, we assess a good manufacturing practice-compliant hollow-fiber bioreactor (Quantum®) for adult chondrocyte manufacture. Chondrocytes were isolated from knee arthroplasty-derived cartilage (n = 5) and expanded in media supplemented with 10% fetal bovine serum (FBS) or 5% human platelet lysate (hPL) on tissue culture plastic (TCP) for a single passage. hPL-supplemented cultures were then expanded in the Quantum bioreactor for a further passage. Matched, parallel cultures in hPL or FBS were maintained on TCP. Chondrocytes from all culture conditions were characterized in terms of growth kinetics, morphology, immunoprofile, chondrogenic potential (chondrocyte pellet assays), and single telomere length analysis. Quantum expansion of chondrocytes resulted in 86.4 ± 38.5 × 106 cells in 8.4 ± 1.5 days, following seeding of 10.2 ± 3.6 × 106 cells. This related to 3.0 ± 1.0 population doublings in the Quantum bioreactor, compared with 2.1 ± 0.6 and 1.3 ± 1.0 on TCP in hPL- and FBS-supplemented media, respectively. Quantum- and TCP-expanded cultures retained equivalent chondropotency and mesenchymal stromal cell marker immunoprofiles, with only the integrin marker, CD49a, decreasing following Quantum expansion. Quantum-expanded chondrocytes demonstrated equivalent chondrogenic potential (as assessed by ability to form and maintain chondrogenic pellets) with matched hPL TCP populations. hPL manufacture, however, led to reduced chondrogenic potential and increased cell surface positivity of integrins CD49b, CD49c, and CD51/61 compared with FBS cultures. Quantum expansion of chondrocytes did not result in shortened 17p telomere length when compared with matched TCP cultures. This study demonstrates that large numbers of adult chondrocytes can be manufactured in the Quantum hollow-fiber bioreactor. This rapid, upscale expansion does not alter chondrocyte phenotype when compared with matched TCP expansion. Therefore, the Quantum provides an attractive method of manufacturing chondrocytes for clinical use. Media supplementation with hPL for chondrocyte expansion may, however, be unfavorable in terms of retaining chondrogenic capacity.

Bio-Enhanced Neoligaments Graft Bearing FE002 Primary Progenitor Tenocytes: Allogeneic Tissue Engineering & Surgical Proofs-of-Concept for Hand Ligament Regenerative Medicine.

Hand tendon/ligament structural ruptures (tears, lacerations) often require surgical reconstruction and grafting, for the restauration of finger mechanical functions. Clinical-grade human primary progenitor tenocytes (FE002 cryopreserved progenitor cell source) have been previously proposed for diversified therapeutic uses within allogeneic tissue engineering and regenerative medicine applications. The aim of this study was to establish bioengineering and surgical proofs-of-concept for an artificial graft (Neoligaments Infinity-Lock 3 device) bearing cultured and viable FE002 primary progenitor tenocytes. Technical optimization and in vitro validation work showed that the combined preparations could be rapidly obtained (dynamic cell seeding of 105 cells/cm of scaffold, 7 days of co-culture). The studied standardized transplants presented homogeneous cellular colonization in vitro (cellular alignment/coating along the scaffold fibers) and other critical functional attributes (tendon extracellular matrix component such as collagen I and aggrecan synthesis/deposition along the scaffold fibers). Notably, major safety- and functionality-related parameters/attributes of the FE002 cells/finished combination products were compiled and set forth (telomerase activity, adhesion and biological coating potentials). A two-part human cadaveric study enabled to establish clinical protocols for hand ligament cell-assisted surgery (ligamento-suspension plasty after trapeziectomy, thumb metacarpo-phalangeal ulnar collateral ligamentoplasty). Importantly, the aggregated experimental results clearly confirmed that functional and clinically usable allogeneic cell-scaffold combination products could be rapidly and robustly prepared for bio-enhanced hand ligament reconstruction. Major advantages of the considered bioengineered graft were discussed in light of existing clinical protocols based on autologous tenocyte transplantation. Overall, this study established proofs-of-concept for the translational development of a functional tissue engineering protocol in allogeneic musculoskeletal regenerative medicine, in view of a pilot clinical trial.

Developing Hypoimmunogenic Human iPSC-Derived Oligodendrocyte Progenitor Cells as an Off-The-Shelf Cell Therapy for Myelin Disorders.

Demyelinating disorders are among the most common and debilitating diseases in neurology. Canavan disease (CD) is a lethal demyelinating disease caused by mutation of the aspartoacylase (ASPA) gene, which leads to the accumulation of its substrate N-acetyl-l-aspartate (NAA), and consequently demyelination and vacuolation in the brain. In this study, hypoimmunogenic human induced pluripotent stem cell (iPSC)-derived oligodendrocyte progenitor cells (OPC) are developed from a healthy donor as an "off-the-shelf" cell therapy. Hypoimmunogenic iPSCs are generated through CRISPR/Cas9 editing of the human leukocyte antigen (HLA) molecules in healthy donor-derived iPSCs and differentiated into OPCs. The OPCs are engrafted into the brains of CD (nur7) mice and exhibit widespread distribution in the brain. The engrafted OPCs mature into oligodendrocytes that express the endogenous wildtype ASPA gene. Consequently, the transplanted mice exhibit elevated human ASPA expression and enzymatic activity and reduced NAA level in the brain. The transplanted OPCs are able to rescue major pathological features of CD, including defective myelination, extensive vacuolation, and motor function deficits. Moreover, the hypoimmunogenic OPCs exhibit low immunogenicity both in vitro and in vivo. The hypoimmunogenic OPCs can be used as "off-the-shelf" universal donor cells to treat various CD patients and many other demyelinating disorders, especially autoimmune demyelinating diseases, such as multiple sclerosis.

Danish phase II trial using adipose tissue derived mesenchymal stromal cells for patients with ischaemic heart failure.

AIMS: Patients suffering from chronic ischaemic heart failure with reduced left ventricular ejection fraction (HFrEF) have reduced quality-of-life, repetitive hospital admissions, and reduced life expectancy. Allogeneic cell therapy is currently investigated as a potential treatment option after initially encouraging results from clinical autologous and allogeneic trials in patients with HFrEF. We aimed to investigate the allogeneic Cardiology Stem Cell Centre Adipose tissue derived mesenchymal Stromal Cell product (CSCC_ASC) as an add-on therapy in patients with chronic HFrEF. METHODS AND RESULTS: This is a Danish multi-centre double-blinded placebo-controlled phase II study with direct intra-myocardial injections of allogeneic CSCC_ASC. A total of 81 HFrEF patients were included and randomized 2:1 to CSCC_ASC or placebo injections. The inclusion criteria were reduced left ventricular ejection fraction (LVEF ≤ 45%), New York Heart Association (NYHA) class II-III despite optimal anti-congestive heart failure medication and no further revascularization options. Injections of 0.3 mL CSCC_ASC (total cell dose 100 × 106 ASCs) (n = 54) or isotonic saline (n = 27) were performed into the viable myocardium in the border zone of infarcted tissue using the NOGA Myostar® catheter (Biological Delivery System, Cordis, Johnson & Johnson, USA). The primary endpoint, left ventricular end systolic volume (LVESV), was evaluated at 6-month follow-up. The safety was measured during a 3-years follow-up period. RESULTS: Mean age was 67.0 ± 9.0 years and 66.6 ± 8.1 years in the ASC and placebo groups, respectively. LVESV was unchanged from baseline to 6-month follow-up in the ASC (125.7 ± 68.8 mL and 126.3 ± 72.5 mL, P = 0.827) and placebo (134.6 ± 45.8 mL and 135.3 ± 49.6 mL, P = 0.855) group without any differences between the groups (0.0 mL (95% CI -9.1 to 9.0 mL, P = 0.992). Neither were there significant changes in left ventricular end diastolic volume or LVEF within the two groups or between groups -5.7 mL (95% CI -16.7 to 5.3 mL, P = 0.306) and -1.7% (95% CI -4.4. to 1.0, P = 0.212), respectively). NYHA classification and 6-min walk test did not alter significantly in the two groups (P > 0.05). The quality-of-life, total symptom, and overall summary score improved significantly only in the ASC group but not between groups. There were 24 serious adverse events (SAEs) in the ASC group and 11 SAEs in the placebo group without any significant differences between the two groups at 1-year follow-up. Kaplan-Meier plot using log-rank test of combined cardiac events showed an overall mean time to event of 30 ± 2 months in the ASC group and 29 ± 2 months in the placebo group without any differences between the groups during the 3 years follow-up period (P = 0.994). CONCLUSIONS: Intramyocardial CSCC_ASC injections in patients with chronic HFrEF were safe but did not improve myocardial function or structure, nor clinical symptoms.

Mucosal-associated invariant T cells for cancer immunotherapy.

Human mucosal-associated invariant T (MAIT) cells are characterized by their expression of an invariant TCR α chain Vα7.2-Jα33/Jα20/Jα12 paired with a restricted TCR ß chain. MAIT cells recognize microbial peptides presented by the highly conserved MHC class I-like molecule MR1 and bridge the innate and acquired immune systems to mediate augmented immune responses. Upon activation, MAIT cells rapidly proliferate, produce a variety of cytokines and cytotoxic molecules, and trigger efficient antitumor immunity. Administration of a representative MAIT cell ligand 5-OP-RU effectively activates MAIT cells and enhances their antitumor capacity. In this review, we introduce MAIT cell biology and their importance in antitumor immunity, summarize the current development of peripheral blood mononuclear cell-derived and stem cell-derived MAIT cell products for cancer treatment, and discuss the potential of genetic engineering of MAIT cells for off-the-shelf cancer immunotherapy.

Comparing the Benefits and Drawbacks of Stem Cell Therapy Based on the Cell Origin or Manipulation Process: Addressing Immunogenicity.

Mesenchymal stem cells (MSCs) are effective in treating autoimmune diseases and managing various conditions, such as engraftment of allogeneic islets. Additionally, autologous and HLA-matched allogeneic MSCs can aid in the engraftment of human allogeneic kidneys with or without low doses of tacrolimus, respectively. However, HLA alloantigens are problematic because cell therapy uses more HLA-mismatched allogeneic cells than autologous for convenience and standardization. In particular, HLA-mismatched MSCs showed increased Ag-specific T/B cells and reduced viability faster than HLA-matched MSCs. In CRISPR/Cas9-based cell therapy, Cas9 induce T cell activation in the recipient's immune system. Interestingly, despite their immunogenicity being limited to the cells with foreign Ags, the accumulation of HLA alloantigen-sensitized T/B cells may lead to allograft rejection, suggesting that alloantigens may have a greater scope of adverse effects than foreign Ags. To avoid alloantigen recognition, the ß2-microglobulin knockout (B2MKO) system, eliminating class-I MHC, was able to avoid rejection by alloreactive CD8 T cells compared to controls. Moreover, universal donor cells in which both B2M and Class II MHC transactivator (CIITA) were knocked out was more effective in avoiding immune rejection than single KO. However, B2MKO and CIITA KO system remain to be controlled and validated for adverse effects such as the development of tumorigenicity due to deficient Ag recognition by CD8 T and CD4 T cells, respectively. Overall, better HLA-matching or depletion of HLA alloantigens prior to cell therapy can reduce repetitive transplantation through the long-term survival of allogeneic cell therapy, which may be especially important for patients seeking allogeneic transplantation.

Human γδ T Cell Subsets and Their Clinical Applications for Cancer Immunotherapy.

Gamma delta (γδ) T cells are a minor population of T cells that share adaptive and innate immune properties. In contrast to MHC-restricted alpha beta (αß) T cells, γδ T cells are activated in an MHC-independent manner, making them ideal candidates for developing allogeneic, off-the-shelf cell-based immunotherapies. As the field of cancer immunotherapy progresses rapidly, different subsets of γδ T cells have been explored. In addition, γδ T cells can be engineered using different gene editing technologies that augment their tumor recognition abilities and antitumor functions. In this review, we outline the unique features of different subsets of human γδ T cells and their antitumor properties. We also summarize the past and the ongoing pre-clinical studies and clinical trials utilizing γδ T cell-based cancer immunotherapy.

HIPGEN: a randomized, multicentre phase III study using intramuscular PLacenta-eXpanded stromal cells therapy for recovery following hip fracture arthroplasty : a study design.

AIMS: The aim of the HIPGEN consortium is to develop the first cell therapy product for hip fracture patients using PLacental-eXpanded (PLX-PAD) stromal cells. METHODS: HIPGEN is a multicentre, multinational, randomized, double-blind, placebo-controlled trial. A total of 240 patients aged 60 to 90 years with low-energy femoral neck fractures (FNF) will be allocated to two arms and receive an intramuscular injection of either 150 × 106 PLX-PAD cells or placebo into the medial gluteal muscle after direct lateral implantation of total or hemi hip arthroplasty. Patients will be followed for two years. The primary endpoint is the Short Physical Performance Battery (SPPB) at week 26. Secondary and exploratory endpoints include morphological parameters (lean body mass), functional parameters (abduction and handgrip strength, symmetry in gait, weightbearing), all-cause mortality rate and patient-reported outcome measures (Lower Limb Measure, EuroQol five-dimension questionnaire). Immunological biomarker and in vitro studies will be performed to analyze the PLX-PAD mechanism of action. A sample size of 240 subjects was calculated providing 88% power for the detection of a 1 SPPB point treatment effect for a two-sided test with an α level of 5%. CONCLUSION: The HIPGEN study assesses the efficacy, safety, and tolerability of intramuscular PLX-PAD administration for the treatment of muscle injury following arthroplasty for hip fracture. It is the first phase III study to investigate the effect of an allogeneic cell therapy on improved mobilization after hip fracture, an aspect which is in sore need of addressing for the improvement in standard of care treatment for patients with FNF. Cite this article: Bone Jt Open 2022;3(4):340-347.

Cell Culture Process Scale-Up Challenges for Commercial-Scale Manufacturing of Allogeneic Pluripotent Stem Cell Products.

Allogeneic cell therapy products, such as therapeutic cells derived from pluripotent stem cells (PSCs), have amazing potential to treat a wide variety of diseases and vast numbers of patients globally. However, there are various challenges related to manufacturing PSCs in single-use bioreactors, particularly at larger volumetric scales. This manuscript addresses these challenges and presents potential solutions to alleviate the anticipated bottlenecks for commercial-scale manufacturing of high-quality therapeutic cells derived from PSCs.

Clinical Study of Mesenchymal Stem/Stromal Cell Therapy for the Treatment of Frailty: A Proposed Experimental Design for Therapeutic and Mechanistic Investigation.

Frailty, a specific condition of increased vulnerability and reduced general health associated with aging in older people, is an emerging problem worldwide with major implications for clinical practice and public health. Recent preclinical and clinical studies have supported the safety of mesenchymal stem/stromal cells (MSCs) in the treatment of frailty. Comprehensive study is needed to assess the interrelationship between the condition of frailty and the effects of MSC-based therapy. This randomized controlled phase I/II trial aims to investigate the safety and potential therapeutic efficacy of the allogeneic administration of umbilical cord-derived MSCs (UC-MSCs) in combination with the standard treatment for frailty in Vietnam. Moreover, this study describes the rationales, study designs, methodologies, and analytical strategies currently employed in stem cell research and clinical studies. The primary outcome measures will include the incidences of prespecified administration-associated adverse events and serious adverse events. The potential efficacy will be evaluated based on improvements in frailty conditions (including those determined through a physical examination, patient-reported outcomes, quality of life, immune markers of frailty, metabolism analysis, and cytokine markers from patient plasma). This clinical trial and stem cell analysis associated with patient sampling at different time points aim to identify and characterize the potential effects of UC-MSCs on improving frailty based on the stem cell quality, cytokine/growth factor secretion profiles of UC-MSCs, cellular senescence, and metabolic analysis of patient CD3+ cells providing fundamental knowledge for designing and implementing research strategies in future studies. Clinical Trials Registration Number: NCT04919135.

Human Mesenchymal Stromal Cells Enhance Cartilage Healing in a Murine Joint Surface Injury Model.

Human umbilical cord (hUC)- or bone marrow (hBM)-derived mesenchymal stromal cells (MSCs) were evaluated as an allogeneic source of cells for cartilage repair. We aimed to determine if they could enhance healing of chondral defects with or without the recruitment of endogenous cells. hMSCs were applied into a focal joint surface injury in knees of adult mice expressing tdTomato fluorescent protein in cells descending from Gdf5-expressing embryonic joint interzone cells. Three experimental groups were used: (i) hUC-MSCs, (ii) hBM-MSCs and (iii) PBS (vehicle) without cells. Cartilage repair was assessed after 8 weeks and tdTomato-expressing cells were detected by immunostaining. Plasma levels of pro-inflammatory mediators and other markers were measured by electrochemiluminescence. Both hUC-MSC (n = 14, p = 0.009) and hBM-MSC (n = 13, p = 0.006) treatment groups had significantly improved cartilage repair compared to controls (n = 18). While hMSCs were not detectable in the repair tissue at 8 weeks post-implantation, increased endogenous Gdf5-lineage cells were detected in repair tissue of hUC-MSC-treated mice. This xenogeneic study indicates that hMSCs enhance intrinsic cartilage repair mechanisms in mice. Hence, hMSCs, particularly the more proliferative hUC-MSCs, could represent an attractive allogeneic cell population for treating patients with chondral defects and perhaps prevent the onset and progression of osteoarthritis.

Engineering Tolerance toward Allogeneic CAR-T Cells by Regulation of MHC Surface Expression with Human Herpes Virus-8 Proteins.

Allogeneic, off-the-shelf (OTS) chimeric antigen receptor (CAR) cell therapies have the potential to reduce manufacturing costs and variability while providing broader accessibility to cancer patients and those with other diseases. However, host-versus-graft reactivity can limit the durability and efficacy of OTS cell therapies requiring new strategies to evade adaptive and innate-immune responses. Human herpes virus-8 (HHV8) maintains infection, in part, by evading host T and natural killer (NK) cell attack. The viral K3 gene encodes a membrane-tethered E3 ubiquitin ligase that discretely targets major histocompatibility complex (MHC) class I components, whereas K5 encodes a similar E3 ligase with broader specificity, including MHC-II and the MHC-like MHC class I polypeptide-related sequence A (MIC-A)- and sequence B (MIC-B)-activating ligands of NK cells. We created γ-retroviruses encoding K3 and/or K5 transgenes that efficiently transduce primary human T cells. Expression of K3 or K5 resulted in dramatic downregulation of MHC-IA (human leukocyte antigen [HLA]-A, -B, and -C) and MHC class II (HLA-DR) cell-surface expression. K3 expression was sufficient for T cells to resist exogenously loaded peptide-MHC-specific cytotoxicity, as well as recognition in one-way allogeneic mixed lymphocyte reactions. Further, in immunodeficient mice engrafted with allogeneic T cells, K3-transduced T cells selectively expanded in vivo. Ectopic K5 expression in MHC class I-, MIC-A+/B+ K562 cells also reduced targeting by primary NK cells. Coexpression of K3 in prostate stem cell antigen (PSCA)-directed, inducible MyD88/CD40 (iMC)-enhanced CAR-T cells did not impact cytotoxicity, T cell growth, or cytokine production against HPAC pancreatic tumor target cells, whereas K5-expressing cells showed a modest reduction in interleukin (IL)-2 production without effect on cytotoxicity. Together, these results support application of these E3 ligases to advance development of OTS CAR-T cell products.

Human serum albumin and chromatin condensation rescue ex vivo expanded γδ T cells from the effects of cryopreservation.

Clinical applications of gamma delta (γδ) T cells have advanced from initial interest in expanding γδ T cells in vivo to the development of a manufacturing process for the ex vivo expansion. To develop an "off-the-shelf" allogeneic γδ T cell product, the cell manufacturing process must be optimized to include cryopreservation. It is known that cryopreservation can dramatically reduce viability of primary cells and other cell types after thawing, although the exact effects of cryopreservation on γδ T cell health and functionality have not yet been characterized. Our aim was to characterize the effects of a freeze/thaw cycle on γδ T cells and to develop an optimized protocol for cryopreservation. γδ T cells were expanded under serum-free conditions, using a good manufacturing practice (GMP) compliant protocol developed by our lab. We observed that cryopreservation reduced cell survival and increased the percentage of apoptotic cells, two measures that could not be improved through the use of 5 GMP compliant freezing media. The choice of thawing medium, specifically human albumin (HSA), improved γδ T cell viability and in addition, chromatin condensation prior to freezing increased cell viability after thawing, which could not be further improved with the use of a general caspase inhibitor. Finally, we found that cryopreserved cells had depolarized mitochondrial membranes and reduced cytotoxicity when tested against a range of leukemia cell lines. These studies provide a detailed analysis of the effects of cryopreservation on γδ T cells and provide methods for improving viability in the post-thaw period.

Microcarrier expansion of c-MycERTAM -modified human olfactory mucosa cells for neural regeneration.

Human olfactory mucosa cells (hOMCs) have potential as a regenerative therapy for spinal cord injury. In our earlier work, we derived PA5 cells, a polyclonal population that retains functional attributes of primary human OMCs. Microcarrier suspension culture is an alternative to planar two-dimensinal culture to produce cells in quantities that can meet the needs of clinical development. This study aimed to screen the effects of 10 microcarriers on PA5 hOMCs yield and phenotype. Studies performed in well plates led to a 2.9-fold higher cell yield on plastic compared to plastic plus microcarriers with upregulation of neural markers ß-III tubulin and nestin for both conditions. Microcarrier suspension culture resulted in concentrations of 1.4 × 105 cells/ml and 4.9 × 104 cells/ml for plastic and plastic plus, respectively, after 7 days. p75NTR transcript was significantly upregulated for PA5 hOMCs grown on Plastic Plus compared to Plastic. Furthermore, coculture of PA5 hOMCs grown on Plastic Plus with a neuronal cell line (NG108-15) led to increased neurite outgrowth. This study shows successful expansion of PA5 cells using suspension culture on microcarriers, and it reveals competing effects of microcarriers on cell expansion versus functional attributes, showing that designing scalable bioprocesses should not only be driven by cell yields.

Novel therapies using cell sheets engineered from allogeneic mesenchymal stem/stromal cells.

Mesenchymal stem/stromal cells (MSCs) have long been recognized to help regenerate tissues, by exploiting their intrinsic potentials for differentiation and secretion of therapeutic paracrine factors together with feasibility for cell banking. These unique MSC properties are attractive to provide effective new cell-based therapies for unmet medical needs. Currently, the infusion of suspended MSCs is accepted as a promising therapy to treat systemic inflammatory diseases. However, low cell engraftment/retention in target organs and off-target entrapment using conventional cell infusion must be improved to provide reliable localized disease treatments. Cell sheet technology offers an alternative: three-dimensional (3D) tissue-like structures can be harvested from culture using mild temperature reduction, and transplanted directly onto target tissue sites without suturing, yielding stable cell engraftment and prolonged cell retention in situ without off-target losses. Engineered MSC sheets directly address two major cell therapy strategies based on their therapeutic benefits: (1) tissue replacements based on mult-ilineage differentiation capacities, focusing on cartilage regeneration in this review, and (2) enhancement of tissue recovery via paracrine signaling, employing their various secreted cytokines to promote neovascularization. MSCs also have production benefits as a promising allogeneic cell source by exploiting their reliable proliferative capacity to facilitate expansion and sustainable cell banking for off-the-shelf therapies. This article reviews the advantages of both MSCs as allogeneic cell sources in contrast with autologous cell sources, and allogeneic MSC sheets engineered on thermo-responsive cell dishes as determined in basic studies and clinical achievements, indicating promise to provide robust new cell therapies to future patients.