Transduction Efficiency Research Articles

Mechanical Disruption of the Inner Limiting Membrane In Vivo Enhances Targeting to the Inner Retina.

To evaluate the effects of mechanical disruption of the inner limiting membrane (ILM) on the ability to target interventions to the inner neurosensory retina in a rodent model. Our study used an animal model to gain insight into the normal physiology of the ILM and advances our understanding of the effects of mechanical ILM removal on the viral transduction of retinal ganglion cells and retinal ganglion cell transplantation. The ILM in the in vivo rat eye was disrupted using mechanical forces applied to the vitreoretinal interface. Immunohistology and electron microscopy were used to verify the removal of the ILM in retina flatmounts and sections. To assess the degree to which ILM disruption enhanced transvitreal access to the retina, in vivo studies involving intravitreal injections of adeno-associated virus (AAV) to transduce retinal ganglion cells (RGCs) and ex vivo studies involving co-culture of human stem cell-derived RGCs (hRGCs) on retinal explants were performed. RGC transduction efficiency and transplanted hRGC integration with retinal explants were evaluated by immunohistology of the retinas. Mechanical disruption of the ILM in the rodent eye was sufficient to remove the ILM from targeted retinal areas while preserving the underlying retinal nerve fiber layer and RGCs. Removal of the ILM enhanced the transduction efficiency of intravitreally delivered AAV threefold (1380.0 ± 290.1 vs. 442.0 ± 249.3 cells/mm2; N = 6; P = 0.034). Removal of the ILM was also sufficient to promote integration of transplanted RGCs within the inner retina. The ILM is a barrier to transvitreally delivered agents including viral vectors and cells. Mechanical removal of the ILM is sufficient to enhance access to the inner retina, improve viral transduction efficiencies of RGCs, and enhance cellular integration of transplanted RGCs with the retina.

Engineering of CD34+ progenitor-derived natural killer cells with higher-affinity CD16a for enhanced antibody-dependent cellular cytotoxicity

Background aimsNatural killer (NK) cell transfer is a promising cellular immunotherapy for cancer. Previously, we developed a robust method to generate large NK cell numbers from CD34+ hematopoietic stem and progenitor cells (HSPCs), which exhibit strong anti-tumor activity. However, since these cells express low levels of the Fc receptor CD16a in vitro, antibody-dependent cellular cytotoxicity (ADCC) by these cells is limited. To broaden clinical applicability of our HSPC-NK cells toward less NK-sensitive malignancies, we aimed to improve ADCC through CD16a transduction. MethodsUsing wildtype and S197P mutant greater-affinity (both with V158) CD16a retroviral transgenes (i.e., a cleavable and noncleavable CD16a upon stimulation), we generated CD16a HSPC-transduced NK cells, with CD34+ cells isolated from umbilical cord blood (UCB) or peripheral blood after G-CSF stem cell mobilization (MPB). CD16a expressing NK cells were enriched using flow cytometry–based cell sorting. Subsequently, phenotypic analyses and functional assays were performed to investigate natural cytotoxicity and ADCC activity. ResultsMean transduction efficiency was 34% for UCB-derived HSPCs and 20% for MPB-derived HSPCs, which was enriched by flow cytometry-based cell sorting to >90% for both conditions. Expression of the transgene remained stable during the entire NK expansion cell generation process. Proliferation and differentiation of HSPCs were not hampered by the transduction process, resulting in effectively differentiated CD56+ NK cells after 5 weeks. Activation of the HSPC-derived NK cells resulted in significant shedding of wildtype CD16a transcribed from the endogenous gene, but not of the noncleavable mutant CD16a protein expressed from the transduced construct. The mean increase of CD107+IFNγ+ expressing NK cells after inducing ADCC was tenfold in enriched noncleavable CD16a HSPC-NK cells. Killing capacity of CD16a-transduced NK cells was significantly improved after addition of a tumor-targeting antibody in tumor cell lines and primary B-cell leukemia and lymphoma cells compared to unmodified HSPC-NK cells. ConclusionsTogether, these data demonstrate that the applicability of adoptive NK cell immunotherapy may be broadened to less NK-sensitive malignancies by upregulation of CD16a expression in combination with the use of tumor-targeting monoclonal antibodies.

Synthesis and properties of antimony sulfoiodide doped with tin (II) cations

Antimony sulfoiodide (SbSI) is a highly efficient energy conversion piezoelectric material. We obtained SbSI doped with tin (II) ions according to the formula Sb[Formula: see text]SnxSI[Formula: see text] ([Formula: see text]–0.1). This heterovalent doping has been performed by the novel method of synthesis in an aqueous solution. The introduction of tin cations leads to a material Curie point increase of more than 10[Formula: see text]K. The samples containing 5[Formula: see text]mol.% of the dopant possess the best piezoelectric properties: piezomodule [Formula: see text][Formula: see text]pC/N, dielectric constant at a frequency of 1[Formula: see text]kHz — 1758, dielectric loss tangent — 0.054, and piezosensitivity — 50[Formula: see text]mV[Formula: see text]×[Formula: see text]m/N. Thus, tin-doped SbSI is a promising material for highly efficient electromechanical transducers and sensors.

Rapid characterization of adeno-associated virus (AAV) capsid proteins using microchip ZipChip CE-MS.

Adeno-associated viruses (AAVs) are viral vectors used as delivery systems for gene therapies. Intact protein characterization of AAV viral capsid proteins (VPs) and their post-translational modifications is critical to ensuring product quality. In this study, microchip-based ZipChip capillary electrophoresis-mass spectrometry (CE-MS) was applied for the rapid characterization of AAV intact VPs, specifically full and empty viral capsids of serotypes AAV6, AAV8 and AAV9, which was accomplished using 5min of analysis time. Low levels of dimethyl sulfoxide (4%) in the background electrolyte (BGE) improved MS signal quality and component detection. A sensitivity evaluation revealed consistent detection of VP proteoforms when as little as 2.64 × 106 viral particles (≈26.4 picograms) were injected. Besides the traditional VP proteoforms used for serotype identification, multiple VP3 variants were detected, including truncated VP3 variants most likely generated by leaky scanning as well as unacetylated and un-cleaved VP3 proteoforms. Phosphorylation, known to impact AAV transduction efficiency, was also seen in all serotypes analysed. Additionally, low abundant fragments originating from either N- or C-terminus truncation were detected. As the aforementioned VP components can impact product quality and efficacy, the ZipChip's ability to rapidly characterize them illustrates its strength in monitoring product quality during AAV production.

Advances in Multi‐Terminal Transistors as Reconfigurable Interconnections for Neuromorphic Sensing and Processing

AbstractInspired by the brain, neuromorphic computing seeks to improve data‐centric applications in terms of power consumption and device footprint. The processing efficiency can be attributed to reconfigurable connections coupled with complex dynamics and efficient data sensing and signal processing. With field‐effect tunability, the three‐terminal thin‐film transistor is initially explored as a switch. Recently, transistors with added gates and modes have been fashioned as multi‐terminal devices capable of interconnecting complex networks and transducing multiple inputs. Here, the advances in both the semiconducting channel material and the configurational design of multi‐terminal devices are reviewed. The use of strong coupling and dynamical material properties in novel ion‐based capacitors, mixed‐ionic‐electronic semiconductors, low‐dimensional confined systems, stimuli‐responsive materials and redox‐active semiconductors has enabled new neuromorphic functionalities. These include improving reconfigurability using the regulatory ability of homeostasis, dendritic integration of sensory signals and the synaptic competition of resources. The transistor devices are also highly relevant in circuits that improve the efficiency of sensory transduction and signal processing; stimuli‐responsive semiconductors that adapt to the sensory inputs can be employed in the transistors and highly efficient sensorimotor connections between sensors and actuators can be emulated. These highly efficient, low‐complexity analog devices are crucial design elements for next‐generation neuromorphic systems.

Comprehensive Comparison of AAV Purification Methods: Iodixanol Gradient Centrifugation vs. Immuno-Affinity Chromatography.

Recombinant adeno-associated viruses (AAVs) have emerged as a widely used gene delivery platform for both basic research and human gene therapy. To ensure and improve the safety profile of AAV vectors, substantial efforts have been dedicated to the vector production process development using suspension HEK293 cells. Here, we studied and compared two downstream purification methods, iodixanol gradient ultracentrifugation versus immuno-affinity chromatography (POROS™ CaptureSelect™ AAVX column). We tested multiple vector batches that were separately produced (including AAV5, AAV8, and AAV9 serotypes). To account for batch-to-batch variability, each batch was halved for subsequent purification by either iodixanol gradient centrifugation or affinity chromatography. In parallel, purified vectors were characterized, and transduction was compared both in vitro and in vivo in mice (using multiple transgenes: Gaussia luciferase, eGFP, and human factor IX). Each purification method was found to have its own advantages and disadvantages regarding purity, viral genome (vg) recovery, and relative empty particle content. Differences in transduction efficiency were found to reflect batch-to-batch variability rather than disparities between the two purification methods, which were similarly capable of yielding potent AAV vectors.

AAV capsid bioengineering in primary human retina models

Adeno-associated viral (AAV) vector-mediated retinal gene therapy is an active field of both pre-clinical as well as clinical research. As with other gene therapy clinical targets, novel bioengineered AAV variants developed by directed evolution or rational design to possess unique desirable properties, are entering retinal gene therapy translational programs. However, it is becoming increasingly evident that predictive preclinical models are required to develop and functionally validate these novel AAVs prior to clinical studies. To investigate if, and to what extent, primary retinal explant culture could be used for AAV capsid development, this study performed a large high-throughput screen of 51 existing AAV capsids in primary human retina explants and other models of the human retina. Furthermore, we applied transgene expression-based directed evolution to develop novel capsids for more efficient transduction of primary human retina cells and compared the top variants to the strongest existing benchmarks identified in the screening described above. A direct side-by-side comparison of the newly developed capsids in four different in vitro and ex vivo model systems of the human retina allowed us to identify novel AAV variants capable of high transgene expression in primary human retina cells.

Bleomycin promotes rAAV2 transduction via DNA-PKcs/Artemis-mediated DNA break repair pathways

Because it is safe and has a simple genome, recombinant adeno-associated virus (rAAV) is an extremely appealing vector for delivery in in vivo gene therapy. However, its low transduction efficiency for some cells, limits its further application in the field of gene therapy. Bleomycin is a chemotherapeutic agent approved by the FDA whose effect on rAAV transduction has not been studied. In this study, we systematically investigated the effect of Bleomycin on the second-strand synthesis and used CRISPR/CAS9 and RNAi methods to understand the effects of Bleomycin on rAAV vector transduction, particularly the effect of DNA repair enzymes. The results showed that Bleomycin could promote rAAV2 transduction both in vivo and in vitro. Increased transduction was discovered to be a direct result of decreased cytoplasmic rAAV particle degradation and increased second-strand synthesis. TDP1, PNKP, and SETMAR are required to repair the DNA damage gap caused by Bleomycin, TDP1, PNKP, and SETMAR promote rAAV second-strand synthesis. Bleomycin induced DNA-PKcs phosphorylation and phosphorylated DNA-PKcs and Artemis promoted second-strand synthesis. The current study identifies an effective method for increasing the capability and scope of in-vivo and in-vitro rAAV applications, which can amplify cell transduction at Bleomycin concentrations. It also supplies information on combining tumor gene therapy with chemotherapy.

Fluorescence of the Retinal Chromophore in Microbial and Animal Rhodopsins.

Fluorescence of the vast majority of natural opsin-based photoactive proteins is extremely low, in accordance with their functions that depend on efficient transduction of absorbed light energy. However, several recently proposed classes of engineered rhodopsins with enhanced fluorescence, along with the discovery of a new natural highly fluorescent rhodopsin, NeoR, opened a way to exploit these transmembrane proteins as fluorescent sensors and draw more attention to studies on this untypical rhodopsin property. Here, we review the available data on the fluorescence of the retinal chromophore in microbial and animal rhodopsins and their photocycle intermediates, as well as different isomers of the protonated retinal Schiff base in various solvents and the gas phase.

Assessment of the Influence of the Geometrical Shape of the Damper on the Efficiency of an Ultrasonic Operation Piezoelectric Transducer.

The results of a study on the geometric shape of the damper on the efficiency of the ultrasonic piezoelectric transducer are presented. In particular, a damper in the form of a truncated cone is considered, the generatrix of which has an inclination angle α relative to the diameter of the piezoceramic plate. The shape of the damper in the form of a truncated cone is chosen based on the a priori assumption that this helps to increase the path of the wave in the damper material due to numerous reflections in it. A criterion for the efficiency of damper operation is proposed. The optimal (from the point of view of the damper efficiency) value of the angle α was determined theoretically and experimentally. The technology of its production is described. Satisfactory agreement between the results of theoretical and experimental studies was noted.

Immunogenicity and protective efficacy of recombinant adenovirus expressing a novel genotype G2b PEDV spike protein in protecting newborn piglets against PEDV.

Porcine epidemic diarrhea (PED) is a highly infectious and economically significant gastrointestinal disorder that affects pigs of all ages. Preventing and controlling PED is achieved by immunizing sows with vaccines, enabling passive piglet immunization via colostrum. The prevalence of G2b porcine epidemic diarrhea virus (PEDV) continues in China despite the use of commercial vaccines, raising questions regarding current vaccine efficacy and the need for novel vaccine development. Adenovirus serotype 5 (Ad5) has several advantages, including high transduction efficiency, a wide range of host cells, and the ability to infect cells at various stages. In this study, we expressed the immunogenic proteins of spike (S) using an Ad5 vector and generated a PED vaccine candidate by inducing significant humoral immunity. The rAd5-PEDV-S prevented PED-induced weight loss, diarrhea, and intestinal damage in piglets. This novel vaccine candidate strain possesses the potential for use in the pig breeding industry.

Regnase-1 overexpression as a therapeutic approach of Marfan syndrome

Rupture or dissection of thoracic aortic aneurysms is still the leading cause of death for patients diagnosed with Marfan syndrome. Inflammation and matrix digestion regulated by matrix metalloproteases (MMPs) play a major role in the pathological remodeling of the aortic media. Regnase-1 is an endoribonuclease shown to cleave the mRNA of proinflammatory cytokines, such as interleukin-6. Considering the major anti-inflammatory effects of regnase-1, here, we aimed to determine whether adeno-associated virus (AAV)-mediated vascular overexpression of the protein could provide protection from the development and progression of aortic aneurysms in Marfan syndrome. The overexpression of regnase-1 resulted in a marked decrease in inflammatory parameters and elastin degradation in aortic smooth muscle cells invitro. Intravenous injection of a vascular-targeted AAV vector resulted in the efficient transduction of the aortic wall and overexpression of regnase-1 in a murine model of Marfan syndrome, associated with lower circulating levels of proinflammatory cytokines and decreased MMP expression and activity. Regnase-1 overexpression strongly improved elastin architecture in the media and reduced aortic diameter at distinct locations. Therefore, AAV-mediated regnase-1 overexpression may represent a novel gene therapy approach for inhibiting aortic aneurysms in Marfan syndrome.

Therapeutic Efficacy of Prodrug Activator Gene Therapy Using Retroviral Replicating Vectors for Human Ovarian Cancer.

Retroviral replicating vectors (RRV) have exhibited efficient tumor transduction and improved therapeutic benefits in a variety of cancer models. In this study, we validated two RRV created from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), which use different cell receptors for virus entry, in human ovarian cancer (OC) cells. Expression levels of the receptors for AMLV (PiT-2) and GALV (PiT-1) in human OC cell lines (A2780, Caov3, RMG-1, SKOV-3), fibroblasts and HEK293 cells were evaluated using quantitative RT-PCR. In vitro RRV-GFP replication was monitored using flow cytometry, and cytotoxicity quantitated using AlamarBlue assay after 5-fluorocytosine treatment of OC cells transduced with RRV expressing the yeast cytosine deaminase prodrug activator gene. In vivo antitumor effect of RRV-mediated prodrug activator gene therapy was investigated in a SKOV-3 subcutaneous tumor model. Quantitative RT-PCR analysis revealed high expression levels of PiT-2 (AMLV receptor) and PiT-1 (GALV receptor) in the RMG-1 and SKOV3 OC cell lines, compared with their levels in non-malignant cells. In RMG-1 and SKOV3 cells, both RRV showed highly efficient RRV replication and spread leading to over 90% transduction by Days 10-13. Additionally, both RRV that express the yeast cytosine deaminase gene demonstrated effective cell killing of RMG-1 and SKOV-3 cells upon treatment with the prodrug 5-fluorocytosine. Notably, RRV-mediated prodrug activator gene therapy showed significant inhibition of subcutaneous SKOV-3 tumor growth in nude mice. RRV-mediated prodrug activator gene therapy may be used for treating PiT-expressing human OC.

CD90-Targeted Cocal-Pseudotyped Lentivirus As a Robust Platform for Human HSC Gene Therapy

Ex vivo gene delivery and editing in hematopoietic stem and progenitor cells (HSPCs) provide a promising approach for the treatment of many chronic conditions such as hemoglobinopathies, lysosomal storage diseases, and HIV. However, such approaches require highly specialized biomedical infrastructure limiting its accessibility to most patients who could benefit. The development of a targeted in vivo approach to HSPC gene delivery/editing is therefore necessary for the broadened use of gene therapies. The CD34 +CD90 + subset of HSPCs represents a highly enriched population for both short-term and long-term hematopoietic lineage repopulation capacity as seen in the non-human primate (NHP) transplant model. Furthermore, ex vivo gene editing of solely CD34 +CD90 + Human and NHP hematopoietic stem cells (HSCs) was sufficient to produce therapeutic thresholds for sickle cell phenotypic amelioration after transplantation. Thus, the development of novel gene therapy vectors targeting the CD34 +CD90 + HSC population is crucial for more accessible and robust gene therapy platforms. Here we report the development of a novel CD90-targeted Cocal pseudotyped lentivirus system capable of targeted transduction of HSPCs in vitro and in vivo. Additionally, we compare transduction efficiency and specificity of this new technology with the more routinely used VSVG targeting methodology. To produce targeted lentivirus, native targeting of VSVG or Cocal envelope to the LDL-Receptor (LDLR) was knocked out by amino acid substitution. An anti-CD90 single-chain variable fragment (scFv) was introduced onto the mutated capsids using CD8 hinge and ICAM transmembrane domains. Mutagenesis of Cocal envelope LDLR binding motif was performed by Gibson cloning and confirmed via nanopore sequencing. Successful mutated viral production was assessed using hydrodynamic titration methods followed by functional analysis of transduction on CD90 + cell lines. In vitro transduction specificity and efficiency assays were performed on Human GCSF-mobilized CD34 +enriched apheresis samples and analyzed by flow cytometry. Humanized NBSGW mice were treated with targeted-lentiviral particles via intravenous (IV) and intraosseous (IO) injection. Mice were necropsied 4-8 weeks post injection. Transgene expression was analyzed by flow cytometry or luminescence imaging and confirmed by PCR on human-CD34 + FACS-sorted cells from necropsied mice. CD90-Targeted Cocal-pseudotyped lentivirus (Cocal-CD90) transduced CD34 +CD90 + HSCs 2.12-fold (p=.017) more robustly than CD90-targeted VSVG-pseudotyped lentivirus (VSVG-CD90) in vitro. Cocal-CD90 virus also showed significantly greater on-target transduction specificity of CD34 +CD90 + HSCs (p=.0008) compared to untargeted wild-type Cocal-pseudotyped lentivirus (Cocal-WT); but similar specificity to VSVG-CD90 virus (p=.118). Unlike VSVG-CD90 virus, we did not observe decreased transduction efficiency of CD34 +CD90 + HSCs using Cocal-CD90 virus in comparison to Cocal-WT virus. IV and IO injection of both CD90-targeted viruses in humanized mice resulted in transduction of immobilized human HSPCs (CD34 +CD38 -CD133 +) in the bone marrow. Transduced cells maintained multilineage differentiation capacity as seen by transgene expression of T-cell, B-cell, and myeloid compartments in the bone marrow, spleen, and peripheral blood at necropsy. As observed in vitro, Cocal-CD90 treated mice consistently displayed more robust transgene expression than VSVG-CD90 treated mice. Importantly, transduction of off-target organs (e.g., liver and lungs) post-IV injection with targeted virus was not detected by luminescence imaging during 8 weeks of follow-up or at necropsy. We provide the first evidence of targeted engineering of a knockout Cocal envelope using the attenuation of native LDLR tropism coupled with a CD90-scFv fusogen protein. Cocal-CD90 virus has increased specificity for CD90 + transduction compared to Cocal-WT lentivirus, without lowering transduction efficiency of on-target cells. Furthermore, in comparison to targeted VSVG engineering, Cocal engineered virus does not decrease transduction efficiency of HSCs in vitro or in vivo. Coupled with Cocal lentiviruses' resistance to human serum inactivation and low immunogenicity, targeted Cocal viral vectors are more suitable for future in vivo gene delivery platforms than their VSVG counterparts.

Type I Interferon Blockade Enhances Transduction Efficiency and Efficacy of Non-Activated CAR T Cells

Chimeric antigen receptor (CAR) T-cell therapies have revolutionized the treatment of advanced hematologic cancers, showing impressive response rates in pivotal trials. Despite this success, challenges in CAR T-cell adoption persist due to tumor resistance mechanisms, manufacturing failures, and limitations in CAR T-cell persistence and effector function. Strategies that promote T-cell persistence, proliferation, and cytotoxic activity are essential to improving long-term outcomes of CAR T therapy. The most common protocol for CAR T-cell manufacturing involves lentiviral vector transduction of activated T-cells followed by ex vivo culture. However, this approach may drive terminal T-cell differentiation and exhaustion, compromising therapeutic efficacy. In contrast, non-activated CAR T-cell manufacturing has shown promise in enhancing T-cell differentiation and tumor control. Yet, the low transduction efficiency of quiescent T-cells remains a challenge. In this study, we explore the role of type I interferon (IFN)-mediated innate immune responses in limiting CAR T manufacturing efficiency. We hypothesize that blocking these responses using the vaccinia virus-encoded type I interferon-binding protein can enhance transduction efficiency and promote less-differentiated CAR T-cell phenotypes, including naïve and central memory-like differentiation. Our findings demonstrate that lentiviral vector transduction triggers an innate immune response in non-activated T-cells, resulting in reduced transduction efficiency, and altered T-cell differentiation. Inhibiting the anti-viral response via anti-IFN leads to improved T-cell differentiation and exhaustion characteristics. Importantly, anti-IFN exposure supports the enrichment of naive-like T cells: a subset noted for superior clinical efficacy. Anti-IFN also reduces LAG3 expression. Collectively, these findings position anti-IFN as a novel means to enhance CAR T-cell persistence and anti-tumor function. We further investigate the influence of sustained anti-IFN exposure by designing lentiviral transfer plasmids encoding both a CAR and secreted type I interferon-binding protein. These cells showed enhanced cytolytic activity and cytokine production against target cells. Our studies in mice show promising results, suggesting that the protein secretion enhance therapeutic activity against tumors employing sustained type I interferon signaling for immune escape. Overall, targeting the type I interferon response during non-activated CAR T-cell manufacturing offers a potential solution to improve transduction efficiency and enhance CAR T-cell functionality. Given the therapeutic promise of non-activated CAR T cells, our findings have immediate translational relevance for broadening their appeal against cancer. Figure 1. Exposure to anti-IFN increases transduction efficiency and naïve-like differentiation of lentivirus-transduced non-activated T-cells A) Freshly isolated human T-cells were incubated in media with aIFN or vehicle, for one hour at room temperature. The cells were resuspended in complete media and then transduced with lentiviral vector encoding iRFP. Expression of iRFP was evaluated 96 h after transduction by flow cytometry. b, Human T-cells were isolated and prepared as above. At 96 h post-transduction, cells were stained with anti-CD45RO and anti-CCR7 antibodies and samples were evaluated by flow cytometry. Data for A and B are representative of multiple experiments with three independent donors.

Paired Analysis from Patients with Myeloma Elucidates Favorable CAR-T Manufacture and Phenotype, Starting from Mobilized Pre-Transplant Apheresis Vs. Non-Mobilized Apheresis after Four Prior Lines

Introduction B-Cell Maturation Antigen (BCMA) targeting chimeric antigen receptor T-cells (CAR-T) have demonstrated unprecedented efficacy in patients with relapsed and refractory multiple myeloma, approved after 4 or more prior lines of therapy (PLOT). Given the known T-cell dysfunction that develops over the disease trajectory, autologous T-cells harvested after many PLOT may not be as biologically active as those collected earlier. Estimates suggest 93% of stored stem cell products collected from MM patients are never used (Liang et al, 2021). We sought to demonstrate the feasibility of using stored mobilized apheresis products to generate anti-BCMA CAR-T cells. Given the heterogeneity that can exist between MM patients, we evaluated the differences in starting T-cell phenotype, manufacturing characteristics, and CAR-T phenotype and function side by side using paired samples from the same patient, obtained prior to transplant or prior to commercial CAR-T. Methods We identified a cohort of paired samples from: 1) Mobilized unfractionated apheresis obtained prior to ASCT (preASCT); and 2) Apheresis for CAR-T manufacture after ≥4 PLOT (preCAR). The flow cytometry panel used for phenotyping included antibodies against: CD3, CD4, CD8, CCR7, CD45RO, CD39, TIGIT, PD1, LAG3 and TCF7. Second generation anti-aBCMA.4-1BB.CD3z (aBCMA.BBz) CAR-T cells were manufactured from CD3+ T cells negatively selected from either sample using a Pan T cell isolation kit (Miltenyi). Post manufacture flow cytometry was used to characterize immunophenotype and transduction efficiency. Doubling time was calculated using trypan blue exclusion on D+2 to D+5 after transduction. A BCMA-expressing myeloma cell line, MM.1S, was used to stimulate and evaluate cytotoxicity. Results In 10 patients with paired preASCT and preCAR samples, the median time from ASCT to CAR-T infusion was 4.2 years (range 2.5-12.5 years), all patients had received three drug induction regimens, and 50% had >1 line of combination therapy prior to ASCT. PreASCT samples ranged in collection from Nov 2008 - April 2019. All patients were triple class exposed prior to collection for CAR-T and treated with median 6 PLOT (range 4-13). Comparison of the T-cell composition in both apheresis samples, revealed a higher CD4:CD8 ratio in preASCT samples (p=0.006), and a higher proportion of stem central memory/SCM (CCR7+CD45RO-, p<0.05, Figure 1) in both CD4 and CD8 compartments. Additionally, in preASCT samples, patients with only 1 line of induction, exhibited a trend toward a higher proportion of SCM T-cells relative to more heavily treated patients. PreASCT CD4 (p=0.002) and CD8 (p=0.01) T-cells expressed more TCF7 compared with preCAR, however T-cells with an effector phenotype were proportionally higher in preCAR samples across both CD4 and CD8 compartments (CCR7-CD45RO-, p<0.05, Figure 1). PreCAR T-cells express higher levels of checkpoints (CP) PD1 and TIGIT, with dual positive CP more prevalent in CD4+ T-cells. CAR-T cells manufactured from preASCT samples expanded better (p=0.048) and also had increased CD4:CD8 ratios (p=0.011) as compared to preCAR, without significant differences in transduction efficiency, CAR density or viability. After 24h stimulation with BCMA+ MM.1S, CD8+ CAR-T cell phenotype was different between preASCT and preCAR products, with more SCM in preASCT (p=0.004) and more effector memory/EM (CCR7-CD45RO+) in preCAR (p=0.008); however, the killing capacity after 24h was similar for both CAR-T products at different E:T ratios. The CAR-T cells generated from T-cells preCAR (later in the disease course) expressed higher TIGIT, LAG3 and CD39 after stimulation, and more dual (p=0.027) or triple (p=0.027) positive CP than those from preASCT T-cells ( Figure 2). Conclusion This study suggests CAR-T cells manufactured from banked mobilized unfractionated stem cell product expand better during manufacture, yield an equally cytotoxic, less differentiated, and less exhausted product than those manufactured from later timepoints (preCAR samples). Importantly, we demonstrate two major findings: (1) that a possibly more efficacious anti-BCMA CAR-T product can be manufactured from cryopreserved, mobilized apheresis material regardless of storage time and (2) cryopreserved preASCT samples may expand autologous CAR-T cell therapy access to heavily treated patients with poor T cell quality. CLF & JAM contributed equally

Optimization of Lentiviral ß-Globin Vectors in a Forward Orientation to Enhance Therapeutic Effectiveness for Sickle Cell Disease

Sickle cell disease (SCD) is a monogenic hemoglobin (Hgb) disorder characterized by a single pointmutation in the β-globin gene which causes Hgb polymerization under low oxygen tension and sickling of red blood cells (RBCs) which in turn drives severe systemic injury. Gene addition technologies like hematopoietic stem cell (HSC) targeted lentiviral (LV) gene therapy are among the most promising curative strategies for SCD. To expand this approach, improvements in LV titer, transduction efficiency, along with suppression of native sickle-globin, are preferred to generate successful therapeutic outcomes. One such improvement targets LV orientation: the relationship between the DNA encoding the β-globin gene in the LV and the promoter driving healthy β-globin gene expression. We created a forward-oriented β-globin-expression vector with an improved titer that results in more efficient transduction of human CD34+ cells in vitro, in human reconstituting CD34+ cells in xenografted mice, and in rhesus reconstituting CD34+ cells(Uchida N. et al., 2019). We report here on further optimization of LVs that in addition to forward orientation, also express shRNA that decreases native β-globin and sickle-globin, which could enhance the assembly of corrected Hgb tetramers. Seven unique constructs which varied in anti-β-globin shRNA sequences, packaging elements, and regulatory elements, were transduced into MEL-BB88 cells and expanded for 2 weeks. gDNA was collected for vector copy number (VCN) analysis, and treated with 10 µM hemin to induce Hgb expression. Five days after hemin-induction, cells were harvested for benzidine staining and immunoblot analysis to validate globin expression. Sca-1+ HSC/progenitor (HSPC) BM cells were isolated from Towne's humanized SCD donor mice prior to stimulation for 48 h with cytokines, then transduced at an MOI of 24 twice, 24 h apart. To assess the in vivo engraftment potential of the transduced HSPCs, 2.5x10^6 cells were transplanted into lethally irradiated CD45.1 recipient mice. Peripheral blood (PB) was sampled at 2-week intervals for PBMC chimerism and lineage engraftment, Hgb electrophoresis, Lorrca OxygenScan and OsmoScan assays, and PB VCN. Whole BM from engrafted recipients was collected at 30 weeks and used for secondary transplantation assays which were again analyzed over 24 weeks for PB engraftment, VCN, and donor Hgb expression. LVs were produced at varying titers and all successfully transduced MEL-BB88 cells with VCNs between 1 and 5 that were maintained following hemin induction. Interestingly, variations in RRE elements dramatically affected human globin expression in vitro. Furthermore, the transplanted mice engrafted fully with HSPCs transduced with multiple vectors. Notably, mice transplanted with HSPCs transduced with vector CX0066p (LCR-Enhancer-P-HBB-shmiR-3g1.223 + 3g2.30a-R1) maintained a VCN of 0.2-0.4 in PB for the 30-week duration of the primary transplant. Cellulose acetate gels of RBCs from PB showed ~40% HbA in a background of HbS. The P50 right-shifted towards normal, RBC counts increased, WBC counts decreased, and OxygenScan parameters were all corrected towards controls and away from those values obtained from mice transplanted with mock transduced HSPCs. EI max and EI min increased while pO2@95% decreased. Lastly, the RBC sickling was corrected in an in vitro sickling assay. In conclusion, based on LV titer, MEL cell expression, transduction efficiency, engraftment of modified cells, in vivo globin expression, anti-sickling activity, and oxygen transport, these constructs provide outstanding Hgb expression and phenotype correction. Consequently, these candidates serve as a solid foundation for future human gene therapy products in the treatment of SCD.

CAR T Cell Exhaustion but Not Ex Vivo Cytotoxicity Is Predictive of Patient Clinical Response: An Interim Analysis of ACIT001/EXC002, a Phase Ib/II Trial of Decentralized Production of CAR T Cells for Treatment of Relapsed/Refractory Aggressive NHL and ALL

Chimeric antigen receptor (CAR) T cell therapy is an increasingly prominent form of next-generation immunotherapy in both hematologic and solid tumor malignancies. Multiple CAR T products have received regulatory approval for use as treatment in relapsed and refractory leukemias and lymphomas. However, approximately half of these patients do not respond to therapy and production of CAR T cells is a difficult, expensive, and time-consuming process. Although much has been learned about the fundamental biology of CAR T cells over the past decade, we are still unable to accurately predict which patients will respond to therapy. In particular, few studies have specifically examined biological characteristics of manufactured CAR T cells and correlated these to relevant clinical outcomes. In this study, we analyzed the lymphocyte populations and CAR T cells from patients enrolled in ACIT001/EXC002, a Canadian multi-centre phase Ib/II single-arm clinical trial of decentralized production of second-generation CD19/41BB/CD3z CAR T cells for treatment of multiply relapsed/refractory non-Hodgkin lymphoma (NHL) and acute lymphoblastic leukemia (ALL). CD3-positive T cells and CAR T products were comprehensively immunophenotyped by flow cytometry. Thecapacity for manufactured CAR T cells to kill tumor cells was assessed using an ex vivo cytotoxicity assay. CAR T cell immunophenotype and cytotoxic potential were correlated with clinical outcomes including response to therapy, progress-free survival (PFS) and incidence of serious adverse events. To date, 23 patients have been enrolled, of which 16 had an aggressive NHL while the remaining 7 had been diagnosed with B cell ALL. Mean age of participants at time of enrollment was 57 (range: 26-77) and 65.2% were male. CAR T product was manufactured using lentiviral transduction with a median transduction efficiency of 32.1% (range: 9.1% to 55.5%). A majority of patients achieved a complete response as evaluated by PET scan on day 28 post-CAR T cell infusion with only 3 patients showing disease progression, 1 patient achieving only partial response, and 3 patients being unevaluable. We were able to evaluate PFS at 6 months in 15 out of 23 patients, with 46.7% of patients experiencing progression and/or mortality within this period. Surprisingly, while CAR T cell cytotoxicity ex vivo trended higher in patients that achieved a complete metabolic response compared to those with partial response or disease progression, this did not reach statistical significance (p = 0.280). However, expression of T cell exhaustion markers such as PD-1 CAR T products was found to be significantly increased in non-responders (p = 0.010). Higher expression of exhaustion markers was also found to be associated with decreased CAR T persistence in vivo as measured by circulating CAR T cell levels at day 14 post-infusion. In our study cohort, CAR T products had either similar or significantly greater proportions of CD4+ as compared to CD8+ T cells. CAR T cells were found to express high levels of HLA-DR and CD127, however this did not correlate with cytotoxicity, response to therapy or PFS. Patient CAR T cells, regardless of the level of exhaustion marker expression, tended to be highly skewed towards the T effector memory subset, with almost no populations of naïve or memory T cells identified. In this analysis of the impact of biological characteristics of CAR T cell products on patient outcomes, we found that CAR T cell exhaustion rather than cytotoxic activity is correlated with response to treatment and higher PFS at 6 months. Interestingly, the degree of patient T cell exhaustion had an impact on transduction efficiency but not the cytotoxic potential of the manufactured CAR T cell product. Taken together, our data indicate that CAR T cell exhaustion and persistence following infusion rather than cytotoxic potential is a key predictor for patient response to treatment. While our conclusions are currently limited by small sample size, more patients are presently being accrued to allow for more robust analysis in the future.

Expression of a Novel Chimeric Inhibitory Receptor Attenuates CD19 CAR T Cells: A Promising Strategy for Toxicity Mitigation

Introduction Chimeric antigen receptor (CAR) T cells targeting CD19 are highly effective in patients with refractory/relapsed acute lymphoblastic leukemia and lymphoma but often trigger severe cytokine release syndrome (sCRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). For r/rALL patients, as many as 77% develop Grade 3-5 CRS and 40% Grade 3-5 ICANS. sCRS occurs when hyperactivated CAR T cells undergo exponential expansion accompanied by the release of excessive amounts of inflammatory cytokines. This leads to a systemic pro-inflammatory state that can cause hypotension, multi-organ failure and even death. Tocilizumab is the only FDA-approved drug for CRS and despite concurrent corticosteroids, often fails to control CRS. Attempts to improve safety have been proposed, but have significant limitations, such as permanent removal of CARs. New and innovative methods to mitigate toxicities are needed. The purpose of this study was to design and test the efficacy of a novel autoregulatory element in the form of a chimeric inhibitory receptor (CIR), that when engaged by a specific CRS-associated pro-inflammatory molecule would trigger inhibitory signaling within CAR T cells. We hypothesize that as cytokine levels increase following activation, these signals can be redirected through responsiveness of the CIR, leading to an attenuation of CAR activity. In this study, we have tested a CIR construct that couples the recognition of IFNγ to the signaling domain of the TIGIT inhibitory receptor. We co-expressed this CIR with a CD19.28ζ CAR to evaluate its ability to attenuate activation and cytokine production and to assess its cytotoxic activity. Methods An anti-IFNγ scFv followed by the transmembrane and intracellular domains of TIGIT were arranged in sequence with the truncated EGFR receptor followed by the CD19.28ζ FMC63 CAR with intervening T2A cleavage domains and cloned into a retroviral backbone. Transduction efficiency was assessed by staining with biotinylated CD19-Fc protein or biotinylated EGFR. Following expansion, memory and effector cell differentiation was evaluated by staining for CD45RO, CD45RA, CCR7, CD127, and CD95. Activation following stimulation with NALM-6 tumor cells was measured by staining for CD69, CD25, CD38, PD-1, and CD107a. Cytotoxicity was evaluated using bioluminescence. To mimic cell types present during CRS, we utilized a coculture assay that consists of CAR T cells, NALM-6 tumor cells, and either macrophages or dendritic cells (DC) differentiated from autologous CD14 + monocytes. Cocultures were incubated for 48 hr. and supernatants were collected for analyses by Luminex. Results CD19 CARs co-expressing a CIR composed of an anti-IFNγ scFv coupled to TIGIT inhibitory signaling expanded during the manufacturing process as well as T cells expressing only CD19 CAR and displayed normal levels of CAR expression. The presence of the CIR did not affect the differentiation profile of memory and effector subsets. CIR-expressing CD19 CARs exhibited robust tumor cell killing of NALM-6 targets equivalent to CD19 CARs. Next, CD19 CAR/CIR cells were pretreated with IFNγ for 24 hr. and then cultured with NALM-6 cells. Exposure to IFNγ resulted in a significant abrogation in cytotoxicity, which was eventually restored with time. Activation profile analysis of CARs equipped with CIR signaling after target stimulation revealed an overall diminution of CARs expressing multiple (4 or 5) activation markers relative to CD19 CARs. Importantly, CIR signaling resulted in significant reductions in the levels of effector, stimulatory, and inflammatory mediators including IFNγ, TNFα, GM-CSF, and IL-17. CARs expressing the CIR exhibited slower killing kinetics when challenged with sequential stimulations by fresh tumor cells. However, in vivo anti-tumor cell activity was not adversely affected as CAR/CIR T cells robustly eliminated disease in leukemia-bearing NSG mice. Lastly, using an in vitro CRS assay, we sought to determine if attenuated CAR T cells would be less potent in activating myeloid cells. We observed that monocyte-derived macrophage and DC activation by CD19 CAR/CIR T cells was reduced as evidenced by decreased secretion of proinflammatory cytokines and chemokines. Conclusion These results provide a compelling proof of concept of a novel self-regulating CAR T cell design with the potential to mitigate severe toxicities in patients.

Anti-BCMA Dual Epitope-Binding CAR-Marrow Infiltrating Lymphocytes (MILs) Could Offer a Potent Innovative Immunotherapeutic Tool Against Multiple Myeloma

Introduction: Chimeric antigen receptor (CAR) T cells are a promising new treatment for multiple myeloma (MM). Marrow-infiltrating lymphocytes (MILs) derived from MM patient's bone marrow are another potential innovative T cell source for CAR-based cell therapy, capable of robustly and specifically targeting myeloma cells through polyclonal targets. MILs possess polyclonal central memory T cell populations, which could be as effective MM killings via polyantigenic cytotoxic T cells with greater recognitions of broad range of tumor antigens via the polyclonal memory TCR as well as the CAR pathway with strong recognition of tumor antigens. Nanobodies are currently the smallest stable antibody molecules having high affinity for antigen, allowing them to strongly penetrate tumor tissue and even directly to the blood-brain barrier while remaining low toxicity and immunogenicity. With the peculiar properties, nanobodies have been widely used as the antigen biding domain of CAR-T cell. In this study, we investigated anti-B-cell maturation antigen (BCMA) dual epitope-binding CAR-MILs against MM. Methods: CD3 + T cells isolated from bone marrow mononuclear cells of MM patients were stimulated with Ex Vivo CD3/CD28 in the presence of recombinant human IL-2. The nanobodies VHH01 and VHH02 were provided as dual epitope-binding for BCMA (Senlang, China). After expansion and activation for 2 days, anti-BCMA dual epitope-binding CAR was transduced to the activated MILs and, they were stimulated with Ex Vivo CD3/CD28 in the presence of recombinant human IL-2, IL-7, IL-15, and IL-21. Expanded and activated BCMA dual epitope-binding CAR-MILs (BCMA CAR-MILs) were harvested on days 7, 14, and 21 for further experiments. Results: The transduction efficiency of BCMA CAR-MILs was approximately 34%, and the expansion of BCMA CAR-MILs was > 100 folds over 21-day culture period. Additionally, BCMA CAR-MILs showed a dramatically increased proportion of CD8 + T cells, while the proportion of CD4 + T cells decreased. Moreover, BCMA CAR-MILs were enriched with a higher proportion of central memory T cells (Tcm) (80%), while the proportions of regulatory T cells (Tregs) and checkpoint molecules (TIGIT, TIM3, and CD73) were not so high. The BCMA CAR-MILs showed potent cytotoxicity, particularly high levels of CD107a degranulation against BCMA-expressing target cancer cell lines, such as Daudi, Raji, RPMI8226, and U266B1 cells, and autologous CD138 + primary myeloma cells. Conclusion: This study suggested that BCMA dual epitope-binding CAR-MILs were easily expanded and activated using bone marrow mononuclear cells from MM patients with high transduction efficiency and induced a potent anti-myeloma immune response, which may offer an alternative innovative immunotherapeutic approach against MM patients in the future.