Correcting inborn errors of immunity: From viral mediated gene addition to gene editing.

Allogeneic hematopoietic stem cell transplantation is an effective treatment to cure inborn errors of immunity. Remarkable progress has been achieved thanks to the development and optimization of effective combination of advanced conditioning regimens and use of immunoablative/suppressive agents preventing rejection as well as graft versus host disease. Despite these tremendous advances, autologous hematopoietic stem/progenitor cell therapy based on ex vivo gene addition exploiting integrating γ-retro- or lenti-viral vectors, has demonstrated to be an innovative and safe therapeutic strategy providing proof of correction without the complications of the allogeneic approach. The recent advent of targeted gene editing able to precisely correct genomic variants in an intended locus of the genome, by introducing deletions, insertions, nucleotide substitutions or introducing a corrective cassette, is emerging in the clinical setting, further extending the therapeutic armamentarium and offering a cure to inherited immune defects not approachable by conventional gene addition. In this review, we will analyze the current state-of-the art of conventional gene therapy and innovative protocols of genome editing in various primary immunodeficiencies, describing preclinical models and clinical data obtained from different trials, highlighting potential advantages and limits of gene correction.

Increased Susceptibility of the CD57- NK Cells Expressing KIR2DL2/3 and NKG2C to iCasp9 Gene Retroviral Transduction and the Relationships with Proliferative Potential, Activation Degree, and Death Induction Response.

Nowadays, the use of genetically modified NK cells is a promising strategy for cancer immunotherapy. The additional insertion of genes capable of inducing cell suicide allows for the timely elimination of the modified NK cells. Different subsets of the heterogenic NK cell population may differ in proliferative potential, in susceptibility to genetic viral transduction, and to the subsequent induction of cell death. The CD57-NKG2C+ NK cells are of special interest as potential candidates for therapeutic usage due to their high proliferative potential and certain features of adaptive NK cells. In this study, CD57- NK cell subsets differing in KIR2DL2/3 and NKG2C expression were transduced with the iCasp9 suicide gene. The highest transduction efficacy was observed in the KIR2DL2/3+NKG2C+ NK cell subset, which demonstrated an increased proliferative potential with prolonged cultivation. The increased transduction efficiency of the cell cultures was associated with the higher expression level of the HLA-DR activation marker. Among the iCasp9-transduced subsets, KIR2DL2/3+ cells had the weakest response to the apoptosis induction by the chemical inductor of dimerization (CID). Thus, KIR2DL2/3+NKG2C+ NK cells showed an increased susceptibility to the iCasp9 retroviral transduction, which was associated with higher proliferative potential and activation status. However, the complete elimination of these cells with CID is impeded.

Reliable RT-qPCR-based titration of retroviral and lentiviral vectors via quantification of residual vector plasmid DNA in samples.

OBJECTIVES: To develop a method for reliable quantification of viral vectors, which is necessary for determining the optimal dose of vector particles in clinical trials to obtain the desired effects without severe unwanted immune responses. RESULTS: A significant level of vector plasmid remained in retroviral and lentiviral vector samples, which led to overestimation of viral titers when using the conventional RT-qPCR-based genomic titration method. To address this problem, we developed a new method in which the residual plasmid was quantified by an additional RT-qPCR step, and standard molecules and primer sets were optimized. The obtained counts were then used to correct the conventionally measured genomic titers of viral samples. While the conventional method produced significantly higher genomic titers for mutant retroviral vectors than for wild-type vectors, our method produced slightly higher or equivalent titers, corresponding with the general idea that mutation of viral components mostly results in reduced or, at best, retained titers. CONCLUSION: Subtraction of the number of residual vector plasmid molecules from the conventionally measured genomic titer can yield reliable quantification of retroviral and lentiviral vector samples, a prerequisite to advancing the safety of gene therapy applications.

Mesenchymal stromal cells retrovirally transduced with prodrug-converting genes are suitable vehicles for cancer gene therapy.

Mesenchymal stem/stromal cells (MSC) possess a set of several fairly unique properties which make them ideally suitable both for cellular therapies and regenerative medicine. These include: relative ease of isolation, the ability to differentiate along mesenchymal and non-mesenchymal lineages in vitro and the ability to be extensively expanded in culture without a loss of differentiative capacity. MSC are not only hypoimmunogenic, but they mediate immunosuppression upon transplantation, and possess pronounced anti-inflammatory properties. They are able to home to damaged tissues, tumors, and metastases following systemic administration. The ability of homing holds big promise for tumor-targeted delivery of therapeutic agents. Viruses are naturally evolved vehicles efficiently transferring their genes into host cells. This ability made them suitable for engineering vector systems for the delivery of genes of interest. MSC can be retrovirally transduced with genes encoding prodrug-converting genes (suicide genes), which are not toxic per se, but catalyze the formation of highly toxic metabolites following the application of a nontoxic prodrug. The homing ability of MSC holds advantages compared to virus vehicles which display many shortcomings in effective delivery of the therapeutic agents. Gene therapies mediated by viruses are limited by their restricted ability to track cancer cells infiltrating into the surrounding tissue, and by their low migratory capacity towards tumor. Thus combination of cellular therapy and gene delivery is an attractive option - it protects the vector from immune surveillance, and supports targeted delivery of a therapeutic gene/protein to the tumor site.

Use of CRISPR/CAS9 Technologies to Study the Role of TLR in Dendritic Cell Subsets.

Dendritic cells (DCs) have a significant role in coordinating both innate and adaptive immunity by serving as sentinels that detect invaders and initiate immune responses to eliminate them, as well as presenting antigens to activate adaptive immune responses that are specific to the antigen and the context in which it was detected. The regulation of DC functions is complex and involves intracellular drivers such as transcription factors and signaling pathways, as well as intercellular interactions with adhesion molecules, chemokines, and their receptors in the microenvironment. Toll-like receptors (TLRs) are crucial for DCs to detect pathogen-associated molecular patterns (PAMPs) and initiate downstream signaling pathways that lead to DC maturation and education in bridging with adaptive immunity, including the upregulation of MHC class II expression, induction of CD80, CD86, and CD40, and production of innate cytokines. Understanding the TLR pathways that DCs use to respond to innate immune stimuli and convert them into adaptive responses is important for new therapeutic targets identification.We present a novel platform that offers a fast and affordable CRISPR-Cas9 screening of genes that are involved in dendritic cells' TLR-dependent activation. Using CRISPR/Cas9 screening to target individual TLR genes in different dendritic cell subsets allows the identification of TLR-dependent pathways that regulate dendritic cell activation and cytokine production. This approach offers the efficient targeting of TLR driver genes to modulate the immune response and identify novel immune response regulators, establishing a causal link between these regulators and functional phenotypes based on genotypes.

Site-specific transgene integration in chimeric antigen receptor (CAR) T cell therapies.

Chimeric antigen receptor (CAR) T cells and natural killer (NK) cells are genetically engineered immune cells that can detect target antigens on the surface of target cells and eliminate them following adoptive transfer. Recent progress in CAR-based therapies has led to outstanding clinical success in certain patients with leukemias and lymphomas and offered therapeutic benefits to those resistant to conventional therapies. The universal approach to stable CAR transgene delivery into the T/NK cells is the use of viral particles. Such approaches mediate semi-random transgene insertions spanning the entire genome with a high preference for integration into sites surrounding highly-expressed genes and active loci. Regardless of the variable CAR expression level based on the integration site of the CAR transgene, foreign integrated DNA fragments may affect the neighboring endogenous genes and chromatin structure and potentially change a transduced T/NK cell behavior and function or even favor cellular transformation. In contrast, site-specific integration of CAR constructs using recent genome-editing technologies could overcome the limitations and disadvantages of universal random gene integration. Herein, we explain random and site-specific integration of CAR transgenes in CAR-T/NK cell therapies. Also, we tend to summarize the methods for site-specific integration as well as the clinical outcomes of certain gene disruptions or enhancements due to CAR transgene integration. Also, the advantages and limitations of using site-specific integration methods are discussed in this review. Ultimately, we will introduce the genomic safe harbor (GSH) standards and suggest some appropriate safety prospects for CAR integration in CAR-T/NK cell therapies.

Development of an in vitro genotoxicity assay to detect retroviral vector-induced lymphoid insertional mutants.

Safety assessment in retroviral vector-mediated gene therapy remains challenging. In clinical trials for different blood and immune disorders, insertional mutagenesis led to myeloid and lymphoid leukemia. We previously developed the In Vitro Immortalization Assay (IVIM) and Surrogate Assay for Genotoxicity Assessment (SAGA) for pre-clinical genotoxicity prediction of integrating vectors. Murine hematopoietic stem and progenitor cells (mHSPCs) transduced with mutagenic vectors acquire a proliferation advantage under limiting dilution (IVIM) and activate stem cell- and cancer-related transcriptional programs (SAGA). However, both assays present an intrinsic myeloid bias due to culture conditions. To detect lymphoid mutants, we differentiated mHSPCs to mature T cells and analyzed their phenotype, insertion site pattern, and gene expression changes after transduction with retroviral vectors. Mutagenic vectors induced a block in differentiation at an early progenitor stage (double-negative 2) compared to fully differentiated untransduced mock cultures. Arrested samples harbored high-risk insertions close to Lmo2, frequently observed in clinical trials with severe adverse events. Lymphoid insertional mutants displayed a unique gene expression signature identified by SAGA. The gene expression-based highly sensitive molecular readout will broaden our understanding of vector-induced oncogenicity and help in pre-clinical prediction of retroviral genotoxicity.

Evaluation of diversity indices to estimate clonal dominance in gene therapy studies.

In cell and gene therapy, achieving the stable engraftment of an abundant and highly polyclonal population of gene-corrected cells is one of the key factors to ensure the successful and safe treatment of patients. Because integrative vectors have been associated with possible risks of insertional mutagenesis leading to clonal dominance, monitoring the relative abundance of individual vector insertion sites in patients' blood cells has become an important safety assessment, particularly in hematopoietic stem cell-based therapies. Clinical studies often express clonal diversity using various metrics. One of the most commonly used is the Shannon index of entropy. However, this index aggregates two distinct aspects of diversity, the number of unique species and their relative abundance. This property hampers the comparison of samples with different richness. This prompted us to reanalyze published datasets and to model the properties of various indices as applied to the evaluation of clonal diversity in gene therapy. A normalized version of the Shannon index, such as Pielou's index, or Simpson's probability index is robust and useful to compare sample evenness between patients and trials. Clinically meaningful standard values for clonal diversity are herein proposed to facilitate the use of vector insertion site analyses in genomic medicine practice.

Selection of Cell Populations with High or Low Surface Marker Expression Using Magnetic Sorting.

Magnetic cell sorting technology stands out because of its speed, simplicity, and ability to process large cell numbers. However, it also suffers from a number of drawbacks, in particular low discrimination power, which results in all-or-none selection outcomes limited to a bulk separation of cell populations into positive and negative fractions, as well as the modest purity of the selected cells and the inability to select subpopulations of cells with high expression of a surface marker. In the present study, we developed a simple solution to this problem and confirmed the effectiveness of this approach by multiple experiments with the magnetic selection of transduced cell populations. Murine NIH 3T3 cells were transduced with the bicistronic retroviral vector constructs co-expressing fluorescent reporter proteins EGFP (enhanced green fluorescent protein) or DsRed-Express 2 and LNGFR (low-affinity nerve growth factor receptor) as surface selection markers. The effects of the magnetic selection of transduced cells with anti-LNGFR Micro Bead (MB) doses ranging from 0.5 to 80 µL have been assessed. Low doses of MBs favored the depletion of weakly positive cells from the population, resulting in the higher expression levels of EGFP or DsRed-Express2 reporters in the selected cell fractions. Low MB doses also contributed to the increased purity of the selected population, even for samples with a low initial percentage of positive cells. At the same time, high MB doses resulted in the increased yield and a more faithful representation of the original expression profiles following selection. We further demonstrate that for populations with fairly narrow distribution of expression levels, it is possible to achieve separation into high- and low-expressing subsets using the two-stage selection scheme based on the sequential use of low and high doses of MBs. For populations with broad expression distribution, a one-stage selection with low or high doses of MBs is sufficient for a clear separation of low- and high-expressing subsets in the column-retained and flow-through fractions, respectively. This study substantially extends the potential of magnetic cell sorting, and may open new possibilities in a number of biomedical applications.

Efficient clinical-grade γ-retroviral vector purification by high-speed centrifugation for CAR T cell manufacturing.

γ-Retroviral vectors (γ-RV) are powerful tools for gene therapy applications. Current clinical vectors are produced from stable producer cell lines which require minimal further downstream processing, while purification schemes for γ-RV produced by transient transfection have not been thoroughly investigated. We aimed to develop a method to purify transiently produced γ-RV for early clinical studies. Here, we report a simple one-step purification method by high-speed centrifugation for γ-RV produced by transient transfection for clinical application. High-speed centrifugation enabled the concentration of viral titers in the range of 107-108 TU/mL with >80% overall recovery. Analysis of research-grade concentrated vector revealed sufficient reduction in product- and process-related impurities. Furthermore, product characterization of clinical-grade γ-RV by BioReliance demonstrated two-logs lower impurities per transducing unit compared with regulatory authority-approved stable producer cell line vector for clinical application. In terms of CAR T cell manufacturing, clinical-grade γ-RV produced by transient transfection and purified by high-speed centrifugation was similar to γ-RV produced from a clinical-grade stable producer cell line. This method will be of value for studies using γ-RV to bridge vector supply between early- and late-stage clinical trials.

Generation of Human 293-F Suspension NGFR Knockout Cells Using CRISPR/Cas9 Coupled to Fluorescent Protein Expression.

Suspension cells derived from human embryonic kidney cells (HEK 293) are attractive cell lines for retroviral vector production in gene therapeutic development studies and applications. The low-affinity nerve growth factor receptor (NGFR) is a genetic marker frequently used as a reporter gene in transfer vectors to detect and enrich genetically modified cells. However, the HEK 293 cell line and its derivatives endogenously express the NGFR protein. To eradicate the high background NGFR expression in future retroviral vector packaging cells, we here employed the CRISPR/Cas9 system to generate human suspension 293-F NGFR knockout cells. The expression of a fluorescent protein coupled via a 2A peptide motif to the NGFR targeting Cas9 endonuclease enabled the simultaneous depletion of cells expressing Cas9 and remaining NGFR-positive cells. Thus, a pure population of NGFR-negative 293-F cells lacking persistent Cas9 expression was obtained in a simple and easily applicable procedure.

Development and clinical translation of ex vivo gene therapy.

Retroviral gene therapy has emerged as a promising therapeutic modality for multiple inherited and acquired human diseases. The capability of delivering curative treatment or mediating therapeutic benefits for a long-term period following a single application fundamentally distinguishes this medical intervention from traditional medicine and various lentiviral/γ-retroviral vector-mediated gene therapy products have been approved for clinical use. Continued advances in retroviral vector engineering, genomic editing, synthetic biology and immunology will broaden the medical applications of gene therapy and improve the efficacy and safety of the treatments based on genetic correction and alteration. This review will summarize the advent and clinical translation of ex vivo gene therapy, with the focus on the milestones during the exploitation of genetically engineered hematopoietic stem cells (HSCs) tackling a variety of pathological conditions which led to marketing approval. Finally, current statue and future prospects of gene editing as an alternative therapeutic approach are also discussed.

Forming megakaryocytes from murine-induced pluripotent stem cells by the inducible overexpression of supporting factors.

BACKGROUND: Platelets are small anucleate cells that circulate in the blood in a resting state but can be activated by external cues. In case of need, platelets from blood donors can be transfused. As an alternative source, platelets can be produced from induced pluripotent stem cells (iPSCs); however, recovered numbers are low. OBJECTIVES: To optimize megakaryocyte (MK) and platelet output from murine iPSCs, we investigated overexpression of the transcription factors GATA-binding factor 1 (GATA1); nuclear factor, erythroid 2; and pre-B-cell leukemia transcription factor 1 (Pbx1) and a hyperactive variant of the small guanosine triphosphatase RhoA (RhoAhc). METHODS: To avoid off-target effects, we generated iPSCs carrying the reverse tetracycline-responsive transactivator M2 (rtTA-M2) in the Rosa26 locus and expressed the factors from Tet-inducible gammaretroviral vectors. Differentiation of iPSCs was initiated by embryoid body (EB) formation. After EB dissociation, early hematopoietic progenitors were enriched and cocultivated on OP9 feeder cells with thrombopoietin and stem cell factor to induce megakaryocyte (MK) differentiation. RESULTS: Overexpression of GATA1 and Pbx1 increased MK output 2- to 2.5-fold and allowed prolonged collection of MK. Cytologic and ultrastructural analyses identified typical MK with enlarged cells, multilobulated nuclei, granule structures, and an internal membrane system. However, GATA1 and Pbx1 expression did not improve MK maturation or platelet release, although in vitro-generated platelets were functional in spreading on fibrinogen or collagen-related peptide. CONCLUSION: We demonstrate that the use of rtTA-M2 transgenic iPSCs transduced with Tet-inducible retroviral vectors allowed for gene expression at later time points during differentiation. With this strategy we could identify factors that increased in vitro MK production.

The Promise and the Hope of Gene Therapy.

It has been over 30 years since visionary scientists came up with the term "Gene Therapy," suggesting that for certain indications, mostly monogenic diseases, substitution of the missing or mutated gene with the normal allele via gene addition could provide long-lasting therapeutic effect to the affected patients and consequently improve their quality of life. This notion has recently become a reality for certain diseases such as hemoglobinopathies and immunodeficiencies and other monogenic diseases. However, the therapeutic wave of gene therapies was not only applied in this context but was more broadly employed to treat cancer with the advent of CAR-T cell therapies. This review will summarize the gradual advent of gene therapies from bench to bedside with a main focus on hemopoietic stem cell gene therapy and genome editing and will provide some useful insights into the future of genetic therapies and their gradual integration in the everyday clinical practice.

Improved functionality and potency of next generation BinMLV viral vectors toward safer gene therapy.

To develop safer retroviral murine leukemia virus (MLV)-based vectors, we previously mutated and re-engineered the MLV integrase: the W390A mutation abolished the interaction with its cellular tethering factors, BET proteins, and a retargeting peptide (the chromodomain of the CBX1 protein) was fused C-terminally. The resulting BET-independent MLVW390A-CBX was shown to integrate efficiently and more randomly, away from typical retroviral markers. In this study, we assessed the functionality and stability of expression of the redistributed MLVW390A-CBX vector in more depth, and evaluated safety using a clinically more relevant vector design encompassing a self-inactivated (SIN) LTR and a weak internal elongation factor 1α short (EFS) promoter. MLVW390A-CBX-EFS produced like MLVWT and efficiently transduced laboratory cells and primary human CD34+ hematopoetic stem cells (HSC) without transgene silencing over time, while displaying a more preferred, redistributed, and safer integration pattern. In a human mesoangioblast (MAB) stem cell model, the myogenic fusion capacity was hindered following MLVWT transduction, while this remained unaffected when applying MLVW390A-CBX. Likewise, smooth muscle cell differentiation of MABs was unaltered by MLVW390A-CBX-EFS. Taken together, our results underscore the potential of MLVW390A-CBX-EFS as a clinically relevant viral vector for ex-vivo gene therapy, combining efficient production with a preferable integration site distribution profile and stable expression over time.

Bcl-2-Assisted Reprogramming of Mouse Astrocytes and Human Fibroblasts into Induced Neurons.

Direct neuronal reprogramming is a promising strategy to generate various types of neurons that are, otherwise, inaccessible for researchers. However, the efficiency of neuronal conversion is highly dependent on the transcription factor used, the identity of the initial cells to convert, their species' background, and the neuronal subtype to which cells will convert. Regardless of these conditioning factors, the apoptotic regulator Bcl-2 acts as a pan-neuronal reprogramming enhancer. Bcl-2 mediates its effect in reprogramming by preventing an overshot of oxidative stress during the acquisition of a neuronal oxidative metabolism, thus reducing cell death by ferroptosis and facilitating the phenotypic conversion. In this chapter, we outline two methods to obtain either mouse or human neurons derived from postnatal astrocytes and skin fibroblasts, respectively. The overall reprogramming strategy is based on the co-expression of Bcl-2 and the transcription factor Neurog2 that produces mostly excitatory neurons. However, the method can be easily adapted to achieve alternative neuronal subtypes by using additional transcription factors, such as Isl1 for motor neurons. Therefore, our approaches provide solid but flexible platforms to obtain human and mouse induced neurons in vitro that can be applied to basic or translational research.

Telomerase Reverse Transcriptase Increases Proliferation and Lifespan of Human NK Cells without Immortalization.

NK cells are the first line of defense against viruses and malignant cells, and their natural functionality makes these cells a promising candidate for cancer cell therapy. The genetic modifications of NK cells, allowing them to overcome some of their inherent limitations, such as low proliferative potential, can enable their use as a therapeutic product. We demonstrate that hTERT-engineered NK cell cultures maintain a high percentage of cells in the S/G2 phase for an extended time after transduction, while the life span of NK cells is measurably extended. Bulk and clonal NK cell cultures pre-activated in vitro with IL-2 and K562-mbIL21 feeder cells can be transduced with hTERT more efficiently compared with the cells activated with IL-2 alone. Overexpressed hTERT was functionally active in transduced NK cells, which displayed upregulated expression of the activation marker HLA-DR, and decreased expression of the maturation marker CD57 and activating receptor NKp46. Larger numbers of KIR2DL2/3+ cells in hTERT-engineered populations may indicate that NK cells with this phenotype are more susceptible to transduction. The hTERT-modified NK cells demonstrated a high natural cytotoxic response towards K562 cells and stably expressed Ki67, a proliferation marker. Overall, our data show that ectopic hTERT expression in NK cells enhances their activation and proliferation, extends in vitro life span, and can be a useful tool in developing NK-based cancer cell therapies.

Efficient Pseudotyping of Different Retroviral Vectors Using a Novel, Codon-Optimized Gene for Chimeric GALV Envelope.

The Gibbon Ape Leukemia Virus envelope protein (GALV-Env) mediates efficient transduction of human cells, particularly primary B and T lymphocytes, and is therefore of great interest in gene therapy. Using internal domains from murine leukemia viruses (MLV), chimeric GALV-Env proteins such as GALV-C4070A were derived, which allow pseudotyping of lentiviral vectors. In order to improve expression efficiency and vector titers, we developed a codon-optimized (co) variant of GALV-C4070A (coGALV-Env). We found that coGALV-Env mediated efficient pseudotyping not only of γ-retroviral and lentiviral vectors, but also α-retroviral vectors. The obtained titers on HEK293T cells were equal to those with the classical GALV-Env, whereas the required plasmid amounts for transient vector production were significantly lower, namely, 20 ng coGALV-Env plasmid per 106 293T producer cells. Importantly, coGALV-Env-pseudotyped γ- and α-retroviral, as well as lentiviral vectors, mediated efficient transduction of primary human T cells. We propose that the novel chimeric coGALV-Env gene will be very useful for the efficient production of high-titer vector preparations, e.g., to equip human T cells with novel specificities using transgenic TCRs or CARs. The considerably lower amount of plasmid needed might also result in a significant cost advantage for good manufacturing practice (GMP) vector production based on transient transfection.

Microfluidic Approach for Highly Efficient Viral Transduction.

Lentiviral vectors enable gene transfer into target cells, but manufacturing is complex, scale-limited, and costly. Here, we describe the use of microfluidic devices for efficient ex vivo gene transfer. Up to four- to fivefold reductions in viral vector usage and two- to fourfold reductions in transduction times can be obtained by using this method.

Ebola Virus Entry: From Molecular Characterization to Drug Discovery.

Ebola Virus Disease (EVD) is one of the most lethal transmissible infections, characterized by a high fatality rate, and caused by a member of the Filoviridae family. The recent large outbreak of EVD in Western Africa (2013⁻2016) highlighted the worldwide threat represented by the disease and its impact on global public health and the economy. The development of highly needed anti-Ebola virus antivirals has been so far hampered by the shortage of tools to study their life cycle in vitro, allowing to screen for potential active compounds outside a biosafety level-4 (BSL-4) containment. Importantly, the development of surrogate models to study Ebola virus entry in a BSL-2 setting, such as viral pseudotypes and Ebola virus-like particles, tremendously boosted both our knowledge of the viral life cycle and the identification of promising antiviral compounds interfering with viral entry. In this context, the combination of such surrogate systems with large-scale small molecule compounds and haploid genetic screenings, as well as rational drug design and drug repurposing approaches will prove priceless in our quest for the development of a treatment for EVD.