Autologous patient-derived exhausted nano T-cells exploit tumor immune evasion to engage an effective cancer therapy.

BACKGROUND: Active targeting by surface-modified nanoplatforms enables a more precise and elevated accumulation of nanoparticles within the tumor, thereby enhancing drug delivery and efficacy for a successful cancer treatment. However, surface functionalization involves complex procedures that increase costs and timelines, presenting challenges for clinical implementation. Biomimetic nanoparticles (BNPs) have emerged as unique drug delivery platforms that overcome the limitations of actively targeted nanoparticles. Nevertheless, BNPs coated with unmodified cells show reduced functionalities such as specific tumor targeting, decreasing the therapeutic efficacy. Those challenges can be overcome by engineering non-patient-derived cells for BNP coating, but these are complex and cost-effective approaches that hinder their wider clinical application. Here we present an immune-driven strategy to improve nanotherapeutic delivery to tumors. Our unique perspective harnesses T-cell exhaustion and tumor immune evasion to develop a groundbreaking new class of BNPs crafted from exhausted T-cells (NExT) of triple-negative breast cancer (TNBC) patients by specific culture methods without sophisticated engineering. METHODS: NExT were generated by coating PLGA (poly(lactic-co-glycolic acid)) nanoparticles with TNBC-derived T-cells exhausted in vitro by acute activation. Physicochemical characterization of NExT was made by dynamic light scattering, electrophoretic light scattering and transmission electron microscopy, and preservation and orientation of immune checkpoint receptors by flow cytometry. The efficacy of chemotherapy-loaded NExT was assessed in TNBC cell lines in vitro. In vivo toxicity was made in CD1 mice. Biodistribution and therapeutic activity of NExT were determined in cell-line- and autologous patient-derived xenografts in immunodeficient mice. RESULTS: We report a cost-effective approach with a good performance that provides NExT naturally endowed with immune checkpoint receptors (PD1, LAG3, TIM3), augmenting specific tumor targeting by engaging cognate ligands, enhancing the therapeutic efficacy of chemotherapy, and disrupting the PD1/PDL1 axis in an immunotherapy-like way. Autologous patient-derived NExT revealed exceptional intratumor accumulation, heightened chemotherapeutic index and efficiency, and targeted the tumor stroma in a PDL1+ patient-derived xenograft model of triple-negative breast cancer. CONCLUSIONS: These advantages underline the potential of autologous patient-derived NExT to revolutionize tailored adoptive cancer nanotherapy and chemoimmunotherapy, which endorses their widespread clinical application of autologous patient-derived NExT.

Estradiol and Estrone Have Different Biological Functions to Induce NF-κB-Driven Inflammation, EMT and Stemness in ER+ Cancer Cells.

In general, the risk of being diagnosed with cancer increases with age; however, the development of estrogen-receptor-positive (ER+) cancer types in women are more closely related to menopausal status than age. In fact, the general risk factors for cancer development, such as obesity-induced inflammation, show differences in their association with ER+ cancer risk in pre- and postmenopausal women. Here, we tested the role of the principal estrogens in the bloodstream before and after menopause, estradiol (E2) and estrone (E1), respectively, on inflammation, epithelial-to-mesenchymal transition (EMT) and cancer stem cell enrichment in the human ER+ cervical cancer cell line HeLa. Our results demonstrate that E1, contrary to E2, is pro-inflammatory, increases embryonic stem-transcription factors (ES-TFs) expression and induces EMT in ER+ HeLa cells. Moreover, we observed that high intratumoural expression levels of 17ß-Hydroxysteroid dehydrogenase (HSD17B) isoforms involved in E1 synthesis is a poor prognosis factor, while overexpression of E2-synthetizing HSD17B isoforms is associated with a better outcome, for patients diagnosed with ER+ ovarian and uterine corpus carcinomas. This work demonstrates that E1 and E2 have different biological functions in ER+ gynaecologic cancers. These results open a new line of research in the study of ER+ cancer subtypes, highlighting the potential key oncogenic role of E1 and HSD17B E1-synthesizing enzymes in the development and progression of these diseases.

Absence of WASp Enhances Hematopoietic and Megakaryocytic Differentiation in a Human Embryonic Stem Cell Model.

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency caused by mutations in the WAS gene and characterized by severe thrombocytopenia. Although the role of WASp in terminally differentiated lymphocytes and myeloid cells is well characterized, its role in early hematopoietic differentiation and in platelets (Plts) biology is poorly understood. In the present manuscript, we have used zinc finger nucleases targeted to the WAS locus for the development of two isogenic WAS knockout (WASKO) human embryonic stem cell lines (hESCs). Upon hematopoietic differentiation, hESCs-WASKO generated increased ratios of CD34(+)CD45(+) progenitors with altered responses to stem cell factor compared to hESCs-WT. When differentiated toward the megakaryocytic linage, hESCs-WASKO produced increased numbers of CD34(+)CD41(+) progenitors, megakaryocytes (MKs), and Plts. hESCs-WASKO-derived MKs and Plts showed altered phenotype as well as defective responses to agonist, mimicking WAS patients MKs and Plts defects. Interestingly, the defects were more evident in WASp-deficient MKs than in WASp-deficient Plts. Importantly, ectopic WAS expression using lentiviral vectors restored normal Plts development and MKs responses. These data validate the AND-1_WASKO cell lines as a human cellular model for basic research and for preclinical studies for WAS.

Mesenchymal stromal cells express GARP/LRRC32 on their surface: effects on their biology and immunomodulatory capacity.

Mesenchymal stromal cells (MSCs) represent a promising tool for therapy in regenerative medicine, transplantation, and autoimmune disease due to their trophic and immunomodulatory activities. However, we are still far from understanding the mechanisms of action of MSCs in these processes. Transforming growth factor (TGF)-ß1 is a pleiotropic cytokine involved in MSC migration, differentiation, and immunomodulation. Recently, glycoprotein A repetitions predominant (GARP) was shown to bind latency-associated peptide (LAP)/TGF-ß1 to the cell surface of activated Foxp3(+) regulatory T cells (Tregs) and megakaryocytes/platelets. In this manuscript, we show that human and mouse MSCs express GARP which presents LAP/TGF-ß1 on their cell surface. Silencing GARP expression in MSCs increased their secretion and activation of TGF-ß1 and reduced their proliferative capacity in a TGF-ß1-independent manner. Importantly, we showed that GARP expression on MSCs contributed to their ability to inhibit T-cell responses in vitro. In summary, we have found that GARP is an essential molecule for MSC biology, regulating their immunomodulatory and proliferative activities. We envision GARP as a new target for improving the therapeutic efficacy of MSCs and also as a novel MSC marker.

Biased and Unbiased Methods for the Detection of Off-Target Cleavage by CRISPR/Cas9: An Overview.

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 endonuclease (Cas9) derived from bacterial adaptive immune systems is a revolutionary tool used in both basic and applied science. It is a versatile system that enables the genome of different species to be modified by generating double strand breaks (DSBs) at specific locations. However, all of the CRISPR/Cas9 systems can also produce DSBs at off-target sites that differ substantially from on-target sites. The generation of DSBs in locations outside the intended site can produce mutations that need to be carefully monitored, especially when using these tools for therapeutic purposes. However, off-target analyses of the CRISPR/Cas9 system have been very challenging, particularly when performed directly in cells. In this manuscript, we review the different strategies developed to identify off-targets generated by CRISPR/cas9 systems and other specific nucleases (ZFNs, TALENs) in real target cells.

Application of Genomic Medicine in Africa: 14th Conference of the African Society of Human Genetics and the 2nd International Congress of the Moroccan Society of Genomics and Human Genetics, Rabat, Morocco 2022.

The 14th African Society of Human Genetics (AfSHG) Morocco Meeting and 2nd International Congress of the Moroccan Society of Genomics and Human Genetics (SM2GH), held in Rabat, Morocco, from December 12 through 17, 2022, brought together 298 attendees from 23 countries, organized by the AfSHG in collaboration with the SM2GH. The conference's overarching theme was "Applications of Genomics Medicine in Africa," covering a wide range of topics, including population genetics, genetics of infectious diseases, hereditary disorders, cancer genetics, and translational genetics. The conference aimed to address the lag in the field of genetics in Africa and highlight the potential for genetic research and personalized medicine on the continent. The goal was to improve the health of African populations and global communities while nurturing the careers of young African scientists in the field. Distinguished scientists from around the world shared their recent findings in genetics, immunogenetics, genomics, genome editing, immunotherapy, and ethics genomics. Precongress activities included a 2-day bioinformatics workshop, "NGS Analysis for Monogenic Disease in African Populations," and a Young Investigators Forum, providing opportunities for young African researchers to showcase their work. The vast genetic diversity of the African continent poses a significant challenge in investigating and characterizing public health issues at the genetic and functional levels. Training, research, and the development of expertise in genetics, immunology, genomics, and bioinformatics are vital for addressing these challenges and advancing genetics in Africa. The AfSHG is committed to leading efforts to enhance genetic research, coordinate training, and foster research collaborations on the continent.

Generating universal anti-CD19 CAR T cells with a defined memory phenotype by CRISPR/Cas9 editing and safety evaluation of the transcriptome.

Introduction: Chimeric antigen receptor-expressing T cells (CAR T cells) have revolutionized cancer treatment, particularly in B cell malignancies. However, the use of autologous T cells for CAR T therapy presents several limitations, including high costs, variable efficacy, and adverse effects linked to cell phenotype. Methods: To overcome these challenges, we developed a strategy to generate universal and safe anti-CD19 CAR T cells with a defined memory phenotype. Our approach utilizes CRISPR/Cas9 technology to target and eliminate the B2M and TRAC genes, reducing graft-versus-host and host-versus-graft responses. Additionally, we selected less differentiated T cells to improve the stability and persistence of the universal CAR T cells. The safety of this method was assessed using our CRISPRroots transcriptome analysis pipeline, which ensures successful gene knockout and the absence of unintended off-target effects on gene expression or transcriptome sequence. Results: In vitro experiments demonstrated the successful generation of functional universal CAR T cells. These cells exhibited potent lytic activity against tumor cells and a reduced cytokine secretion profile. The CRISPRroots analysis confirmed effective gene knockout and no unintended off-target effects, validating it as a pioneering tool for on/off-target and transcriptome analysis in genome editing experiments. Discussion: Our findings establish a robust pipeline for manufacturing safe, universal CAR T cells with a favorable memory phenotype. This approach has the potential to address the current limitations of autologous CAR T cell therapy, offering a more stable and persistent treatment option with reduced adverse effects. The use of CRISPRroots enhances the reliability and safety of gene editing in the development of CAR T cell therapies. Conclusion: We have developed a potent and reliable method for producing universal CAR T cells with a defined memory phenotype, demonstrating both efficacy and safety in vitro. This innovative approach could significantly improve the therapeutic landscape for patients with B cell malignancies.

The Black Hole: CAR T Cell Therapy in AML.

Despite exhaustive studies, researchers have made little progress in the field of adoptive cellular therapies for relapsed/refractory acute myeloid leukemia (AML), unlike the notable uptake for B cell malignancies. Various single antigen-targeting chimeric antigen receptor (CAR) T cell Phase I trials have been established worldwide and have recruited approximately 100 patients. The high heterogeneity at the genetic and molecular levels within and between AML patients resembles a black hole: a great gravitational field that sucks in everything. One must consider the fact that only around 30% of patients show a response; there are, however, consequential off-tumor effects. It is obvious that a new point of view is needed to achieve more promising results. This review first introduces the unique therapeutic challenges of not only CAR T cells but also other adoptive cellular therapies in AML. Next, recent single-cell sequencing data for AML to assess somatically acquired alterations at the DNA, epigenetic, RNA, and protein levels are discussed to give a perspective on cellular heterogeneity, intercellular hierarchies, and the cellular ecosystem. Finally, promising novel strategies are summarized, including more sophisticated next-generation CAR T, TCR-T, and CAR NK therapies; the approaches with which to tailor the microenvironment and target neoantigens; and allogeneic approaches.

Failure of ALL recognition by CAR T cells: a review of CD 19-negative relapses after anti-CD 19 CAR-T treatment in B-ALL.

The use of chimeric antigen receptor (CAR) T lymphocytes in the treatment of refractory or relapsed (R/R) B cell acute lymphoblastic leukemia (B-ALL) has meant a radical change in the prognosis of these patients, whose chances of survival with conventional treatment are very low. The current probability of event-free survival by R/R B-ALL patients treated using anti-CD 19 CART cell therapy is as high as 50-60% at 1.5 years, which is a very important advance for this group of very ill patients. Although most patients (70 to 94%) achieve complete remission (CR), the main problem continues to be relapse of the disease. Most relapses, both in clinical trials and real-world evidence, are due to failure of CAR-T cell expansion or limited CAR-T persistence. However, despite the adequate functioning of infused CART lymphocytes, the tumor cells of an important group of patients manage to evade CAR-T attack, resulting in a CD 19-negative relapse. Several mechanisms have been described that may be able to produce the escape of leukemic cells, such as acquired mutations and alternative splicing of the CD19 antigen, CD19 epitope loss or masking, leukemia lineage switching, and trogocytosis. In the present review, we comprehensively analyze the leukemic cell escape mechanisms, the incidence of CD19-negative relapse reported in clinical trials and real-world evidence (outside clinical trials), and provide an update on the main lines of current research into the prevention of leukemia evasion.

CRISPR delivery with extracellular vesicles: Promises and challenges.

The CRISPR gene editing tool holds great potential for curing genetic disorders. However, the safe, efficient, and specific delivery of the CRISPR/Cas9 components into cells and tissues remains a challenge. While many currently available delivery methods achieve high levels of gene editing effects in vivo, they often result in genotoxicity and immunogenicity. Extracellular vesicles (EVs), which are cell-derived lipid nanoparticles, are capable of transferring protein and nucleic acid cargoes between cells, making them a promising endogenous alternative to synthetic delivery methods. This review provides a comprehensive analysis of the currently available strategies for EV-mediated delivery of CRISPR/Cas9. These strategies include cell-based, passive loading obtained by overexpression of CRISPR/Cas9, active loading involving protein or RNA dimerization, and loading into already purified EVs. All these approaches suggest that EV-based CRISPR/Cas9 delivery is useful for achieving both in vitro and in vivo gene editing. Despite that, substantial variations in cellular uptake and gene editing efficiencies indicate that further improvement and standardization are required for the therapeutic use of EVs as a CRISPR/Cas9 delivery vehicle. These improvements include, but is not limited to, the high-yield purification of EVs, increased loading and release efficiencies, as well as improved tissue- or cell-specific targeting specificities.

High-Density Lipoprotein in Metabolic Disorders and Beyond: An Exciting New World Full of Challenges and Opportunities.

High-density lipoprotein (HDL) is an enigmatic member of the plasma lipid and lipoprotein transport system, best known for its ability to promote the reverse cholesterol efflux and the unloading of excess cholesterol from peripheral tissues. More recently, data in experimental mice and humans suggest that HDL may play important novel roles in other physiological processes associated with various metabolic disorders. Important parameters in the HDL functions are its apolipoprotein and lipid content, further reinforcing the principle that HDL structure defines its functionality. Thus, based on current evidence, low levels of HDL-cholesterol (HDL-C) or dysfunctional HDL particles contribute to the development of metabolic diseases such as morbid obesity, type 2 diabetes mellitus, and nonalcoholic fatty liver disease. Interestingly, low levels of HDL-C and dysfunctional HDL particles are observed in patients with multiple myeloma and other types of cancer. Therefore, adjusting HDL-C levels within the optimal range and improving HDL particle functionality is expected to benefit such pathological conditions. The failure of previous clinical trials testing various HDL-C-raising pharmaceuticals does not preclude a significant role for HDL in the treatment of atherosclerosis and related metabolic disorders. Those trials were designed on the principle of "the more the better", ignoring the U-shape relationship between HDL-C levels and morbidity and mortality. Thus, many of these pharmaceuticals should be retested in appropriately designed clinical trials. Novel gene-editing-based pharmaceuticals aiming at altering the apolipoprotein composition of HDL are expected to revolutionize the treatment strategies, improving the functionality of dysfunctional HDL.

Preclinical Evidence for the Use of Oral Mesenchymal Stem Cell-Derived Extracellular Vesicles in Bone Regenerative Therapy: A Systematic Review.

The development of extracellular vesicles (EVs) therapies has revolutionized personalized medicine, opening up new possibilities for treatment. EVs have emerged as a promising therapeutic tool within this field due to their crucial role in intercellular communication across various cell types and organisms. This systematic review aims to evaluate the therapeutic potential of oral mesenchymal stem cell (MSC)-derived EVs for bone regeneration, specifically focusing on findings from preclinical models. Sixteen articles meeting the inclusion criteria were selected following document analysis. The biological effects of oral MSC-derived EVs predominantly involve the upregulation of proteins associated with angiogenesis, and inflammation resolution, alongside the downregulation of proinflammatory cytokines. Moreover, these therapeutic agents have been found to contain a significant quantity of different molecules (proteins, lipids, DNA, microRNAs, etc) further contributing to their modulatory potential. The findings from this systematic review underscore that oral MSC-derived EVs, irrespective of their specific population, have the ability to enhance the osteogenic repair response in maxillary bone or periodontal defects. In summary, this systematic review highlights the promising potential of oral MSC-derived EVs for bone regeneration based on evidence from preclinical models. The comprehensive assessment of their biological effects and the presence of microRNAs underscores their therapeutic significance. These findings support the utilization of oral MSC-derived EVs in enhancing the osteogenic repair response in various maxillary bone or periodontal defects, providing insights into the mechanisms involved and potential therapeutic applications in the field of personalized medicine.

Lentiviral vectors for inducible, transactivator-free advanced therapy medicinal products: Application to CAR-T cells.

Controlling transgene expression through an externally administered inductor is envisioned as a potent strategy to improve safety and efficacy of gene therapy approaches. Generally, inducible ON systems require a chimeric transcription factor (transactivator) that becomes activated by an inductor, which is not optimal for clinical translation due to their toxicity. We generated previously the first all-in-one, transactivator-free, doxycycline (Dox)-responsive (Lent-On-Plus or LOP) lentiviral vectors (LVs) able to control transgene expression in human stem cells. Here, we have generated new versions of the LOP LVs and have analyzed their applicability for the generation of inducible advanced therapy medicinal products (ATMPs) with special focus on primary human T cells. We have shown that, contrary to all other cell types analyzed, an Is2 insulator must be inserted into the 3' long terminal repeat of the LOP LVs in order to control transgene expression in human primary T cells. Importantly, inducible primary T cells generated by the LOPIs2 LVs are responsive to ultralow doses of Dox and have no changes in phenotype or function compared with untransduced T cells. We validated the LOPIs2 system by generating inducible CAR-T cells that selectively kill CD19+ cells in the presence of Dox. In summary, we describe here the first transactivator-free, all-one-one system capable of generating Dox-inducible ATMPs.

Progress and harmonization of gene editing to treat human diseases: Proceeding of COST Action CA21113 GenE-HumDi.

The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the "Genome Editing to treat Human Diseases" (GenE-HumDi) network, uniting various stakeholders such as pharmaceutical companies, academic institutions, regulatory agencies, biotech firms, and patient advocacy groups. GenE-HumDi's primary objective is to expedite the application of genome editing for therapeutic purposes in treating human diseases. To achieve this goal, GenE-HumDi is organized in several working groups, each focusing on specific aspects. These groups aim to enhance genome editing technologies, assess delivery systems, address safety concerns, promote clinical translation, and develop regulatory guidelines. The network seeks to establish standard procedures and guidelines for these areas to standardize scientific practices and facilitate knowledge sharing. Furthermore, GenE-HumDi aims to communicate its findings to the public in accessible yet rigorous language, emphasizing genome editing's potential to revolutionize the treatment of many human diseases. The inaugural GenE-HumDi meeting, held in Granada, Spain, in March 2023, featured presentations from experts in the field, discussing recent breakthroughs in delivery methods, safety measures, clinical translation, and regulatory aspects related to gene editing.

Physiological lentiviral vectors for the generation of improved CAR-T cells.

Anti-CD19 chimeric antigen receptor (CAR)-T cells have achieved impressive outcomes for the treatment of relapsed and refractory B-lineage neoplasms. However, important limitations still remain due to severe adverse events (i.e., cytokine release syndrome and neuroinflammation) and relapse of 40%-50% of the treated patients. Most CAR-T cells are generated using retroviral vectors with strong promoters that lead to high CAR expression levels, tonic signaling, premature exhaustion, and overstimulation, reducing efficacy and increasing side effects. Here, we show that lentiviral vectors (LVs) expressing the transgene through a WAS gene promoter (AW-LVs) closely mimic the T cell receptor (TCR)/CD3 expression kinetic upon stimulation. These AW-LVs can generate improved CAR-T cells as a consequence of their moderate and TCR-like expression profile. Compared with CAR-T cells generated with human elongation factor α (EF1α)-driven-LVs, AW-CAR-T cells exhibited lower tonic signaling, higher proportion of naive and stem cell memory T cells, less exhausted phenotype, and milder secretion of tumor necrosis factor alpha (TNF-α) and interferon (IFN)-É£ after efficient destruction of CD19+ lymphoma cells, both in vitro and in vivo. Moreover, we also showed their improved efficiency using an in vitro CD19+ pancreatic tumor model. We finally demonstrated the feasibility of large-scale manufacturing of AW-CAR-T cells in guanosine monophosphate (GMP)-like conditions. Based on these data, we propose the use of AW-LVs for the generation of improved CAR-T products.

Rnd3 Expression is Necessary to Maintain Mitochondrial Homeostasis but Dispensable for Autophagy.

Autophagy is a highly conserved process that mediates the targeting and degradation of intracellular components to lysosomes, contributing to the maintenance of cellular homeostasis and to obtaining energy, which ensures viability under stress conditions. Therefore, autophagy defects are common to different neurodegenerative disorders. Rnd3 belongs to the family of Rho GTPases, involved in the regulation of actin cytoskeleton dynamics and important in the modulation of cellular processes such as migration and proliferation. Murine models have shown that Rnd3 is relevant for the correct development and function of the Central Nervous System and lack of its expression produces several motor alterations and neural development impairment. However, little is known about the molecular events through which Rnd3 produces these phenotypes. Interestingly we have observed that Rnd3 deficiency correlates with the appearance of autophagy impairment profiles and irregular mitochondria. In this work, we have explored the impact of Rnd3 loss of expression in mitochondrial function and autophagy, using a Rnd3 KO CRISPR cell model. Rnd3 deficient cells show no alterations in autophagy and mitochondria turnover is not impaired. However, Rnd3 KO cells have an altered mitochondria oxidative metabolism, resembling the effect caused by oxidative stress. In fact, lack of Rnd3 expression makes these cells strictly dependent on glycolysis to obtain energy. Altogether, our results demonstrate that Rnd3 is relevant to maintain mitochondria function, suggesting a possible relationship with neurodegenerative diseases.

Erratum: Physiological lentiviral vectors for the generation of improved CAR-T cells.

[This corrects the article DOI: 10.1016/j.omto.2022.05.003.].

Efficacy and safety of universal (TCRKO) ARI-0001 CAR-T cells for the treatment of B-cell lymphoma.

Autologous T cells expressing the Chimeric Antigen Receptor (CAR) have been approved as advanced therapy medicinal products (ATMPs) against several hematological malignancies. However, the generation of patient-specific CAR-T products delays treatment and precludes standardization. Allogeneic off-the-shelf CAR-T cells are an alternative to simplify this complex and time-consuming process. Here we investigated safety and efficacy of knocking out the TCR molecule in ARI-0001 CAR-T cells, a second generation αCD19 CAR approved by the Spanish Agency of Medicines and Medical Devices (AEMPS) under the Hospital Exemption for treatment of patients older than 25 years with Relapsed/Refractory acute B cell lymphoblastic leukemia (B-ALL). We first analyzed the efficacy and safety issues that arise during disruption of the TCR gene using CRISPR/Cas9. We have shown that edition of TRAC locus in T cells using CRISPR as ribonuleorproteins allows a highly efficient TCR disruption (over 80%) without significant alterations on T cells phenotype and with an increased percentage of energetic mitochondria. However, we also found that efficient TCRKO can lead to on-target large and medium size deletions, indicating a potential safety risk of this procedure that needs monitoring. Importantly, TCR edition of ARI-0001 efficiently prevented allogeneic responses and did not detectably alter their phenotype, while maintaining a similar anti-tumor activity ex vivo and in vivo compared to unedited ARI-0001 CAR-T cells. In summary, we showed here that, although there are still some risks of genotoxicity due to genome editing, disruption of the TCR is a feasible strategy for the generation of functional allogeneic ARI-0001 CAR-T cells. We propose to further validate this protocol for the treatment of patients that do not fit the requirements for standard autologous CAR-T cells administration.

Improved Functionality of Integration-Deficient Lentiviral Vectors (IDLVs) by the Inclusion of IS2 Protein Docks.

Integration-deficient lentiviral vectors (IDLVs) have recently generated increasing interest, not only as a tool for transient gene delivery, but also as a technique for detecting off-target cleavage in gene-editing methodologies which rely on customized endonucleases (ENs). Despite their broad potential applications, the efficacy of IDLVs has historically been limited by low transgene expression and by the reduced sensitivity to detect low-frequency off-target events. We have previously reported that the incorporation of the chimeric sequence element IS2 into the long terminal repeat (LTR) of IDLVs increases gene expression levels, while also reducing the episome yield inside transduced cells. Our study demonstrates that the effectiveness of IDLVs relies on the balance between two parameters which can be modulated by the inclusion of IS2 sequences. In the present study, we explore new IDLV configurations harboring several elements based on IS2 modifications engineered to mediate more efficient transgene expression without affecting the targeted cell load. Of all the insulators and configurations analysed, the insertion of the IS2 into the 3'LTR produced the best results. After demonstrating a DAPI-low nuclear gene repositioning of IS2-containing episomes, we determined whether, in addition to a positive effect on transcription, the IS2 could improve the capture of IDLVs on double strand breaks (DSBs). Thus, DSBs were randomly generated, using the etoposide or locus-specific CRISPR-Cas9. Our results show that the IS2 element improved the efficacy of IDLV DSB detection. Altogether, our data indicate that the insertion of IS2 into the LTR of IDLVs improved, not only their transgene expression levels, but also their ability to be inserted into existing DSBs. This could have significant implications for the development of an unbiased detection tool for off-target cleavage sites from different specific nucleases.

Baboon Envelope Pseudotyped "Nanoblades" Carrying Cas9/gRNA Complexes Allow Efficient Genome Editing in Human T, B, and CD34+ Cells and Knock-in of AAV6-Encoded Donor DNA in CD34+ Cells.

Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into human blood cells can be challenging. Here, we have utilized "nanoblades," a new technology that delivers a genomic cleaving agent into cells. These are modified murine leukemia virus (MLV) or HIV-derived virus-like particle (VLP), in which the viral structural protein Gag has been fused to Cas9. These VLPs are thus loaded with Cas9 protein complexed with the guide RNAs. Highly efficient gene editing was obtained in cell lines, IPS and primary mouse and human cells. Here, we showed that nanoblades were remarkably efficient for entry into human T, B, and hematopoietic stem and progenitor cells (HSPCs) thanks to their surface co-pseudotyping with baboon retroviral and VSV-G envelope glycoproteins. A brief incubation of human T and B cells with nanoblades incorporating two gRNAs resulted in 40 and 15% edited deletion in the Wiskott-Aldrich syndrome (WAS) gene locus, respectively. CD34+ cells (HSPCs) treated with the same nanoblades allowed 30-40% exon 1 drop-out in the WAS gene locus. Importantly, no toxicity was detected upon nanoblade-mediated gene editing of these blood cells. Finally, we also treated HSPCs with nanoblades in combination with a donor-encoding rAAV6 vector resulting in up to 40% of stable expression cassette knock-in into the WAS gene locus. Summarizing, this new technology is simple to implement, shows high flexibility for different targets including primary immune cells of human and murine origin, is relatively inexpensive and therefore gives important prospects for basic and clinical translation in the area of gene therapy.