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Spinal muscular atrophy (SMA) is a rare neuromuscular condition, characterized by loss of motor neurons as a result of a mutation in the survival motor neuron gene. This results in muscle wasting and in the most common and severe type, death before 24 months. Over the recent years there has been a dynamic shift in the therapeutic options for these patients involving both clinical trials in genetic modifying therapies to indirectly improve the survival motor neuron protein level and hence strength, muscle promotor therapies, up/down regulation of modifier genes and more recently gene therapy to replace the mutated survival motor neuron gene. This review addresses the pathogenesis of SMA and the resultant therapeutic approaches as well as the current state in clinical trials and future development.

Gene therapy is a rapidly developing field, and adeno-associated virus (AAV) is a leading viral vector candidate for therapeutic gene delivery. Newly engineered AAVs with improved abilities are now entering the clinic. It has proven challenging, however, to predict the translational potential of gene therapies developed in animal models, due to cross-species differences. Human retinal explants are the only available model of fully developed human retinal tissue, and are thus important for the validation of candidate AAV vectors. In this study, we evaluated 18 wildtype and engineered AAV capsids in human retinal explants using a recently developed single-cell RNA-Seq AAV engineering pipeline (scAAVengr). Human retinal explants retained the same major cell types as fresh retina, with similar expression of cell-specific markers, except for a cone population with altered expression of cone-specific genes. The efficiency and tropism of AAVs in human explants were quantified, with single-cell resolution. The top performing serotypes, K91, K912, and 7m8, were further validated in non-human primate and human retinal explants. Together, this study provides detailed information about the transcriptome profiles of retinal explants, and quantifies the infectivity of leading AAV serotypes in human retina, accelerating the translation of retinal gene therapies to the clinic.Graphic abstract

Extensive clinical data from liver-mediated gene therapy trials have shown that dose-dependent immune responses against the vector capsid may impair or even preclude transgene expression if not managed successfully with prompt immune suppression. The goal of this preclinical study was to generate an adeno-associated viral (AAV) vector capable of expressing therapeutic levels of B-domain deleted factor VIII (FVIII) at the lowest possible vector dose to minimize the potential Risk of a capsid-mediated immune response in the clinical setting. Here, we describe the studies that identified the investigational agent SPK-8011, currently being evaluated in a phase 1/2 study (NCT03003533) in individuals with hemophilia A. In particular, the potency of our second-generation expression cassettes was evaluated in mice and in non-human primates using two different bioengineered capsids (AAV-Spark100 and AAV-Spark200). At 2 weeks after gene transfer, primates transduced with 2 × 1012 vg/kg AAV-Spark100-FVIII or AAV-Spark200-FVIII expressed FVIII antigen levels of 13% ± 2% and 22% ± 6% of normal, respectively. Collectively, these preclinical results validate the feasibility of lowering the AAV capsid dose for a gene-based therapeutic approach for hemophilia A to a dose level orders of magnitude lower than the first-generation vectors in the clinic., Graphical Abstract, The studies presented here represent the proof-of-concept work that generated the investigational agent SPK-8011, currently being evaluated in a phase 1/2 study (NCT03003533) for treatment of hemophilia A. Through a multi-pronged optimization approach, an adeno-associated viral vector capable of expressing therapeutic levels of hFVIII at the lowest possible vector dose was generated.

This study aimed to systematically review and summarize gene therapy treatment for monogenic retinal and optic nerve diseases.This review was prospectively registered (CRD42021229812). A comprehensive literature search was performed in Ovid MEDLINE, Ovid Embase, Cochrane Central, and clinical trial registries (February 2021). Clinical studies describing DNA-based gene therapy treatments for monogenic posterior ocular diseases were eligible for inclusion. Risk of bias evaluation was performed. Data synthesis was undertaken applying Synthesis Without Meta-analysis guidelines.This study identified 47 full-text publications, 50 conference abstracts, and 54 clinical trial registry entries describing DNA-based ocular gene therapy treatments for 16 different genetic variants. Study summaries and visual representations of safety and efficacy outcomes are presented for 20 unique full-text publications in RPE65-mediated retinal dystrophies, choroideremia, Leber hereditary optic neuropathy, rod-cone dystrophy, achromatopsia, and X-linked retinoschisis. The most common adverse events were related to lid/ocular surface/cornea abnormalities in subretinal gene therapy trials and anterior uveitis in intravitreal gene therapy trials.There is a high degree of variability in ocular monogenic gene therapy trials with respect to study design, statistical methodology, and reporting of safety and efficacy outcomes. This review improves the accessibility and transparency in interpreting gene therapy trials to date.

Aromatic L-amino acid decarboxylase deficiency results in decreased neurotransmitter levels and severe motor dysfunction. Twenty-six patients without head control received bilateral intraputaminal infusions of a recombinant adeno-associated virus type 2 vector containing the human aromatic L-amino acid decarboxylase gene (eladocagene exuparvovec) and have completed 1-year evaluations. Rapid improvements in motor and cognitive function occurred within 12 months after gene therapy and were sustained during follow-up for >5 years. An increase in dopamine production was demonstrated by positron emission tomography and neurotransmitter analysis. Patient symptoms (mood, sweating, temperature, and oculogyric crises), patient growth, and patient caretaker quality of life improved. Although improvements were observed in all treated participants, younger age was associated with greater improvement. There were no treatment-associated brain injuries, and most adverse events were related to underlying disease. Post-surgery complications such as cerebrospinal fluid leakage were managed with standard of care. Most patients experienced mild to moderate dyskinesia that resolved in a few months. These observations suggest that eladocagene exuparvovec treatment for aromatic L-amino acid decarboxylase deficiency provides durable and meaningful benefits with a favorable safety profile.

The programmable nuclease technology CRISPR-Cas9 has revolutionized gene editing in the last decade. Due to the risk of off-target editing, accurate and sensitive methods for off-target characterization are crucial prior to applying CRISPR-Cas9 therapeutically. Here, we utilized a rhesus macaque model to compare the predictive values of CIRCLE-seq, an in vitro off-target prediction method, with in silico prediction (ISP) based solely on genomic sequence comparisons. We use AmpliSeq HD error-corrected sequencing to validate off-target sites predicted by CIRCLE-seq and ISP for a CD33 guide RNA (gRNA) with thousands of off-target sites predicted by ISP and CIRCLE-seq. We found poor correlation between the sites predicted by the two methods. When almost 500 sites predicted by each method were analyzed by error-corrected sequencing of hematopoietic cells following transplantation, 19 off-target sites revealed insertion or deletion mutations. Of these sites, 8 were predicted by both methods, 8 by CIRCLE-seq only, and 3 by ISP only. The levels of cells with these off-target edits exhibited no expansion or abnormal behavior in vivo in animals followed for up to 2 years. In addition, we utilized an unbiased method termed CAST-seq to search for translocations between the on-target site and off-target sites present in animals following transplantation, detecting one specific translocation that persisted in blood cells for at least 1 year following transplantation. In conclusion, neither CIRCLE-seq or ISP predicted all sites, and a combination of careful gRNA design, followed by screening for predicted off-target sites in target cells by multiple methods, may be required for optimizing safety of clinical development.

To defense harmful stimuli or maintain the immune homeostasis, the body produces and recruits a superfamily of cytokines such as interleukins, interferons, chemokines etc. Among them, chemokines act as crucial regulators in defense systems. CCL5/CCR5 combination is known for facilitating inflammatory responses, as well as inducing the adhesion and migration of different T cell subsets in immune responses. In addition, recent studies have shown that the interaction between CCL5 and CCR5 is involved in various pathological processes including inflammation, chronic diseases, cancers as well as the infection of COVID-19. This review focuses on how CCL5/CCR5 axis participates in the pathological processes of different diseases and their relevant signaling pathways for the regulation of the axis. Moreover, we highlighted the gene therapy and chemotherapy studies for treating CCR5-related diseases, including the ongoing clinical trials. The barriers and perspectives for future application and translational research were also summarized.

Sertoli cells (SCs) are immune privileged cells found in the testis that function to immunologically protect maturing germ cells from immune destruction. This immune protection is due to the blood-testis-barrier, which prevents infiltration of cytotoxic immune cells and antibodies, and SC production of immunomodulatory factors, that favor a tolerogenic environment. The ability of SCs to create an immune privileged environment has led to the exploration of their potential use in the treatment of various diseases. SCs have been utilized to create a tolerogenic ectopic microenvironment, to protect co-grafted cells, and to deliver therapeutic proteins through gene therapy. To date, numerous studies have reported the potential use of SCs for the treatment of diabetes, neurodegenerative disorders, and restoration of spermatogenesis. Additionally, SCs have been investigated as a delivery vehicle for therapeutic products to treat other diseases like Laron syndrome, muscular dystrophy, and infections. This review will provide an overview of these therapeutic applications.

Stem cell-based therapeutic approach provides a possible treatment for critical limb ischemia (CLI) by inducing revascularization and regenerating ischemic tissue. However, the clinical benefit is modest due to low cell survival and limited efficacy after transplantation. Cardiac-derived stem cells (CSCs) might be a novel cell source for CLI treatment owing to their superb endothelial differentiation potential and angiogenic paracrine functions. In this study, the angiogenic ability of CSCs was maximized by genetic engineering with constitutively active form of hypoxia-inducible factor-1α (CA-HIF-1α), resistant to oxygen-dependent degradation. CSCs transfected with CA-HIF-1α (CA-HIF-CSCs) promoted supplementary expression of proangiogenic factors including VEGF, bFGF, Ang-1 and PDGF-B, along with enhanced angiogenic function including migratory effect, tube formation and endothelial differentiation potential. In the mouse CLI model, CA-HIF-CSCs transplanted into the ischemic region using fibrin gel as cell delivery vehicle, improved blood perfusion and limb functional recovery with minimal incidence of foot necrosis and limb loss by promoting new vessel formation. Histological evidence further confirmed that CA-HIF-CSC/gel treatment markedly alleviated muscle degeneration and fibrosis. CSCs genetically engineered with constitutively active HIF-1α provide a novel therapeutic modality in CLI combining stem cell and gene therapy.

Bone marrow failure syndromes encompass a range of inherited and acquired hematological diseases that result in insufficient blood cell production, which leads to severe complications including anemia, weakening of the immune system, impaired coagulation, and increased risk of cancer. Within inherited bone marrow failure syndromes, a number of genetically distinct diseases have been described including Shwachman-Diamond syndrome and Fanconi anemia. Given the genetic complexity and poor prognosis of these inherited bone marrow failure syndromes, there is increasing interest in both characterizing the genetic landscapes of these diseases and developing novel gene therapies in order to effectively monitor and cure patients. These topics were the focus of the winter 2021 International Society for Experimental Hematology New Investigator Webinar, which featured presentations by Drs. Akiko Shimamura and Paula Rio. Here, we review the topics covered within this webinar.

Background Gliomas are the most common type of central nervous system tumor in adults, and they have an extremely poor prognosis. Gliomas are classified into 4 grades, with low-grade gliomas (LGGs) constituting grades I and II and glioblastoma multiforme (GBM) constituting grade IV. Breast cancer susceptibility genes BRCA1 and BRCA2 play a role in DNA repair and are required for genome stability. Methods We analyzed the LGG and GBM cohorts from The Cancer Genome Atlas. We used Kaplan-Meier and log-rank analysis to determine the relationship between BRCA1 and BRCA2 expression and survival. Results Correlation of BRCA1 and BRCA2 expression with survival in patients with LGG was significant (P = 0.00 and P = 0.00). The higher the levels of expression were, the lower survival rates were in both LGG and GBM cohorts, but the correlation was not significant in patients with GBM (P Conclusions Our findings suggest that BRCA1 and BRCA2 can be regarded as poor prognostic factors in patients with glioma, with greater significance in patients with LGG. In the future, more in-depth experiments will enable us to elucidate the mechanism of gliomagenesis and identify potential gene therapy targets.

Congenital disorders resulting in pathological protein deficiencies are most often treated postnatally with protein or enzyme replacement therapies. However, treatment of these disorders in utero before irreversible disease onset could significantly minimize disease burden, morbidity, and mortality. One possible strategy for the prenatal treatment of congenital disorders is in utero delivery of messenger RNA (mRNA). mRNA is a nucleic acid therapeutic that has previously been investigated as a platform for protein replacement therapies and gene editing technologies. While viral vectors have been explored to induce intracellular expression of mRNA, they are limited in their clinical application due to risks associated with immunogenicity and genomic integration. As an alternative to viral vectors, safe and efficient in utero mRNA delivery can be achieved using ionizable lipid nanoparticles (LNPs). While LNPs have demonstrated potent in vivo mRNA delivery to the liver following intravenous administration, intra-amniotic delivery has the potential to deliver mRNA to cells and tissues beyond those in the liver, such as in the skin, lung, and digestive tract. However, LNP stability in fetal amniotic fluid and how this stability affects mRNA delivery has not been previously investigated. Here, we engineered a library of LNPs using orthogonal design of experiments (DOE) to evaluate how LNP structure affects their stability in amniotic fluid ex utero and whether a lead candidate identified from these stability measurements enables intra-amniotic mRNA delivery in utero. We used a combination of techniques including dynamic light scattering (DLS), transmission electron microscopy (TEM), and chromatography followed by protein content quantification to screen LNP stability in amniotic fluids. These results identified multiple lead LNP formulations that are highly stable in amniotic fluids ranging from small animals to humans, including mouse, sheep, pig, and human amniotic fluid samples. We then demonstrate that stable LNPs from the ex utero screen in mouse amniotic fluid enabled potent mRNA delivery in primary fetal lung fibroblasts and in utero following intra-amniotic injection in a murine model. This exploration of ex utero stability in amniotic fluids demonstrates a means by which to identify novel LNP formulations for prenatal treatment of congenital disorders via in utero mRNA delivery.

Haemophilia A and B are congenital bleeding disorders characterized by missing or defective factor VIII or factor IX, respectively. Factor replacement therapy has been the gold standard for prophylaxis and treatment of bleeding complications. However, the inconvenience of regular intravenous administration, along with progression of arthropathy and development of inhibitors has driven the need for alternative treatment options, such as extended half-life products, non-factor coagulation products, such as subcutaneous emicizumab, blocking natural anticoagulants (rebalancing haemostatic agents) and gene therapy, which have been useful to control bleeding or are currently under late-phase clinical investigation.

Lentiviral vectors (LVs) are robust delivery vehicles for gene therapy as they can efficiently integrate transgenes into host cell genomes. However, LVs with lengthy or complex expression cassettes typically are produced at low titers and have reduced gene transfer capacity, creating barriers for clinical and commercial applications. Modifications of the packaging cell line and methods may be able to produce complex vectors at higher titer and infectivity and may improve production of many different LVs. In this study, we identified two host restriction factors in HEK293T packaging cells that impeded LV production, 2′-5′-oligoadenylate synthetase 1 (OAS1) and low-density lipoprotein receptor (LDLR). Knocking out these two genes separately led to ∼2-fold increases in viral titer. We created a monoclonal cell line, CRISPRed HEK293T to Disrupt Antiviral Response (CHEDAR), by successively knocking out OAS1, LDLR, and PKR, a previously identified factor impeding LV titers. Packaging in CHEDAR yielded ∼7-fold increases in physical particles, full-length vector RNA, and vector titers. In addition, overexpressing transcription elongation factors, SPT4 and SPT5, during packaging improved the production of full-length vector RNA, thereby increasing titers by ∼2-fold. Packaging in CHEDAR with over-expression of SPT4 and SPT5 led to ∼11-fold increases of titers. These approaches improved the production of a variety of LVs, especially vectors with low titers or with internal promoters in the reverse orientation, and may be beneficial for multiple gene therapy applications.

SURF1 (surfeit locus protein 1)-related Leigh syndrome is an early-onset neurodegenerative disorder, characterized by reduction in complex IV activity, resulting in disrupted mitochondrial function. Currently, there are no treatment options available. To test our hypothesis that adeno-associated viral vector serotype 9 (AAV9)/human SURF1 (hSURF1) gene replacement therapy can provide a potentially meaningful and long-term therapeutic benefit, we conducted preclinical efficacy studies using SURF1 knockout mice and safety evaluations with wild-type (WT) mice. Our data indicate that with a single intrathecal (i.t.) administration, our treatment partially and significantly rescued complex IV activity in all tissues tested, including liver, brain, and muscle. Accordingly, complex IV content (examined via MT-CO1 protein expression level) also increased with our treatment. In a separate group of mice, AAV9/hSURF1 mitigated the blood lactic acidosis induced by exhaustive exercise at 9 months post-dosing. A toxicity study in WT mice showed no adverse effects in either the in-life portion or after microscopic examination of major tissues up to a year following the same treatment regimen. Taken together, our data suggest a single dose, i.t. administration of AAV9/hSURF1 is safe and effective in improving biochemical abnormalities induced by SURF1 deficiency with potential applicability for SURF1-related Leigh syndrome patients.

Because current mainstream anti-glycolipid GD2 therapeutics for neuroblastoma (NB) have limitations, such as severe adverse effects, improved therapeutics are needed. In this study, we developed a GD2 aptamer (DB99) and constructed a GD2-aptamer-mediated multifunctional nanomedicine (ANM) with effective, precise, and biocompatible properties, which functioned both as chemotherapy and as gene therapy for NB. DB99 can bind to GD2+ NB tumor cells but has minimal cross-reactivity to GD2− cells. Furthermore, ANM is formulated by self-assembly of synthetic aptamers DB99 and NB-specific MYCN small interfering RNA (siRNA), followed by self-loading of the chemotherapeutic agent doxorubicin (Dox). ANM is capable of specifically recognizing, binding, and internalizing GD2+, but not GD2−, NB tumor cells in vitro. Intracellular delivery of ANM activates Dox release for chemotherapy and MYCN-siRNA-induced MYCN silencing. ANM specifically targets, and selectively accumulates in, the GD2+ tumor site in vivo and further induces growth inhibition of GD2+ tumors in vivo; in addition, ANM generates fewer or no side effects in healthy tissues, resulting in markedly longer survival with fewer adverse effects. These results suggest that the GD2-aptamer-mediated, targeted drug delivery system may have potential applications for precise treatment of NB., Graphical abstract, Multifunctional nanomedicine (ANM), with effective, precise, and biocompatible properties based on a GD2 aptamer, to achieve both chemotherapy and gene therapy of neuroblastoma was constructed by Zhang et al. This GD2-aptamer-mediated, targeted, multifunctional nanomedicine system may open a new avenue and have potential applications for precision treatment of NB.

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is an autosomal recessive disorder that develops in infancy and arises from mutation of the immunoglobulin helicase μ-binding protein 2 (IGHMBP2) gene. Whereas IGHMBP2 is ubiquitously expressed, loss or reduction of function leads to alpha motor neuron loss and skeletal muscle atrophy. We previously developed a gene therapy strategy for SMARD1 using a single-stranded AAV9-IGHMBP2 vector and compared two different delivery methods in a validated SMARD1 mouse model. An important question in the field relates to the temporal requirements for this or any potential treatment. To examine the therapeutic window, we utilized our recently developed SMARD1 model, FVB/NJ-Ighmpb2nmd-2J, to deliver AAV9-IGHMBP2 at four different time points starting at post-natal day 2 (P2) through P8. At each time point, significant improvements were observed in survival, weight gain, and motor function. Similarly, treatment improved important hallmarks of disease, including motor unit pathology. Whereas improvements were more pronounced in the early-treatment groups, even the later-treatment groups displayed significant phenotypic improvements. This work suggests that an effective gene therapy strategy could provide benefits to pre-symptomatic and early-symptomatic individuals, thereby expanding the potential therapeutic window for SMARD1.

With the recent advances in gene editing with systems such as CRISPR-Cas9, precise genome editing in utero is on the horizon. Sickle cell disease is an excellent candidate for in utero fetal gene therapy, because the disease is monogenic, causes irreversible harm, and has life-limiting morbidity. Gene therapy has recently been proven to be effective in an adolescent patient. Several hurdles still impede the progress for fetal gene therapy in humans, including an incomplete understanding of the fetal immune system, unclear maternal immune responses to in utero gene therapy, risks of off-target effects from gene editing, gestational age constraints, and ethical questions surrounding fetal genetic intervention. However, none of these barriers appears insurmountable, and the journey to in utero gene therapy for sickle cell disease and other conditions should be well underway.

Amyotrophic lateral sclerosis (ALS) has historically posed unique challenges for gene-therapy-based approaches, due to a paucity of therapeutic targets as well as the difficulty of accessing both the brain and spinal cord. Recent advances in our understanding of disease mechanism and ALS genetics, however, have combined with tremendous strides in CNS targeting, gene delivery, and gene editing and knockdown techniques to open new horizons of therapeutic possibility. Gene therapy clinical trials are currently underway for ALS patients with SOD1 mutations, C9orf72 hexanucleotide repeat expansions, ATXN2 trinucleotide expansions, and FUS mutations, as well as sporadic disease without known genetic cause. In this review, we provide an in-depth exploration of the state of ALS-directed gene therapy, including antisense oligonucleotides, RNA interference, CRISPR, adeno-associated virus (AAV)-mediated trophic support, and antibody-based methods. We discuss how each of these approaches has been implemented across known genetic causes as well as sporadic ALS, reviewing preclinical studies as well as completed and ongoing human clinical trials. We highlight the transformative potential of these evolving technologies as the gene therapy field advances toward a true disease-modifying treatment for this devastating illness.

Genome editing is potentially a curative technique available to all individuals with β-hemoglobinopathies, including sickle cell disease (SCD). Fetal hemoglobin (HbF) inhibits sickle hemoglobin (HbS) polymerization, and it is well described that naturally occurring hereditary persistence of HbF (HPFH) alleviates disease symptoms; therefore, reawakening of developmentally silenced HbF in adult red blood cells (RBCs) has long been of interest as a therapeutic strategy. Recent advances in genome editing platforms, particularly with the use of CRISPR-Cas9, have paved the way for efficient HbF induction through the creation of artificial HPFH mutations, editing of transcriptional HbF silencers, and modulating epigenetic intermediates that govern HbF expression. Clinical trials investigating BCL11A enhancer editing in patients with β-hemoglobinopathies have demonstrated promising results, although follow-up is short and the number of patients treated to date is low. While practical, economic, and clinical challenges of genome editing are well recognized by the scientific community, potential solutions to overcome these hurdles are in development. Here, we review the recent progress and obstacles yet to be overcome for the most effective and feasible HbF reactivation practice using CRISPR-Cas9 genome editing as a curative strategy for patients with SCD., Graphical abstract, Given the ability of fetal hemoglobin (HbF) to inhibit sickle hemoglobin polymerization, HbF reactivation by the creation of naturally occurring HbF-associated mutations, editing HbF repressors/their binding site, or epigenetic intermediates using CRISPR-Cas9 are promising. Recent clinical data are encouraging; nevertheless, long-term follow-up is lacking, and genome editing safety and efficacy concerns remain.

Adenoviruses (Ads), common self-limiting pathogens in humans and animals, usually cause conjunctivitis, mild upper respiratory tract infection or gastroenteritis in humans and hepatotoxicity syndrome in chickens and dogs, posing great threats to public health and livestock husbandry. Artificially modified Ads, which wipe out virulence-determining genes, are the most frequently used viral vectors in gene therapy, and some Ad vector (AdV)-related medicines and vaccines have been licensed and applied. Inherent liver tropism enables AdVs to specifically deliver drugs/genes to the liver; however, AdVs are closely associated with acute hepatotoxicity in immunocompromised individuals, and the side effects of AdVs, which stimulate a strong inflammatory reaction in the liver and cause acute hepatotoxicity, have largely limited clinical application. Therefore, this review systematically elucidates the intimate relationship between AdVs and hepatotoxicity in terms of virus and host and precisely illustrates the accumulated understanding in this field over the past decades. This review demonstrates the liver tropism of AdVs and molecular mechanism of AdV-induced hepatotoxicity and looks at the studies on AdV-mediated animal hepatotoxicity, which will undoubtedly deepen the understanding of AdV-caused liver injury and be of benefit in the further safe development of AdVs.

Immune responses to adeno-associated virus (AAV) capsids limit the therapeutic potential of AAV gene therapy. Herein, we model clinical immune responses by generating AAV capsid-specific chimeric antigen receptor (AAV-CAR) T cells. We then modulate immune responses to AAV capsid with AAV-CAR regulatory T cells (Tregs). AAV-CAR Tregs in vitro display phenotypical Treg surface marker expression, and functional suppression of effector T cell proliferation and cytotoxicity. In mouse models, AAV-CAR Tregs mediated continued transgene expression from an immunogenic capsid, despite antibody responses, produced immunosuppressive cytokines, and decreased tissue inflammation. AAV-CAR Tregs are also able to bystander suppress immune responses to immunogenic transgenes similarly mediating continued transgene expression, producing immunosuppressive cytokines, and reducing tissue infiltration. Taken together, AAV-CAR T cells and AAV-CAR Tregs are directed and powerful immunosuppressive tools to model and modulate immune responses to AAV capsids and transgenes in the local environment., Graphical abstract, We herein utilized AAV-CAR Tregs to create tolerogenic environments suppressing both capsid and AAV-delivered transgene responses allowing for continued transgene expression from transduced cells. This induced local immune tolerance may ultimately be safer clinically than systemic, broad-spectrum immune suppression.

Delivery of adeno-associated viral vectors (AAVs) to cerebrospinal fluid (CSF) has emerged as a promising approach to achieve widespread transduction of the central nervous system (CNS) and peripheral nervous system (PNS), with direct applicability to the treatment of a wide range of neurological diseases, particularly lysosomal storage diseases. Although studies in small animal models have provided proof of concept and experiments in large animals demonstrated feasibility in bigger brains, there is not much information on long-term safety or durability of the effect. Here, we report a 7-year study in healthy beagle dogs after intra-CSF delivery of a single, clinically relevant dose (2 × 1013 vg/dog) of AAV9 vectors carrying the canine sulfamidase, the enzyme deficient in mucopolysaccharidosis type IIIA. Periodic monitoring of CSF and blood, clinical and neurological evaluations, and magnetic resonance and ultrasound imaging of target organs demonstrated no toxicity related to treatment. AAV9-mediated gene transfer resulted in detection of sulfamidase activity in CSF throughout the study. Analysis at tissue level showed widespread sulfamidase expression and activity in the absence of histological findings in any region of encephalon, spinal cord, or dorsal root ganglia. Altogether, these results provide proof of durability of expression and long-term safety for intra-CSF delivery of AAV-based gene transfer vectors encoding therapeutic proteins to the CNS., Graphical abstract, Seven-year follow-up of dogs after intra-CSF administration of AAV9-sulfamidase vectors results in detection of sulfamidase activity in CSF and widespread transgene expression in CNS, PNS, and liver, in the absence of any adverse events. Proof of durability and safety of this gene therapy support its clinical translation to treat CNS diseases.

Off-target editing is one of the main safety concerns for the use of CRISPR-Cas9 genome editing in gene therapy. These unwanted modifications could lead to malignant transformation, which renders tumorigenicity assessment of gene therapy products indispensable. In this study, we established two in vitro transformation assays, the soft agar colony-forming assay (SACF) and the growth in low attachment assay (GILA) as alternative methods for tumorigenicity evaluation of genome-edited cells. Using a CRISPR-Cas9-based approach to transform immortalized MCF10A cells, we identified PTPN12, a known tumor suppressor, as a valid positive control in GILA and SACF. Next, we measured the limit of detection for both assays and proved that SACF is more sensitive than GILA (0.8% versus 3.1% transformed cells). We further validated SACF and GILA by identifying a set of positive and negative controls and by testing the suitability of another cell line (THLE-2). Moreover, in contrast to SACF and GILA, an in vivo tumorigenicity study failed to detect the known tumorigenic potential of PTPN12 deletion, demonstrating the relevance of GILA and SACF in tumorigenicity testing. In conclusion, SACF and GILA are both attractive and valuable additions to preclinical safety assessment of gene therapy products., Graphical abstract, Anchorage-independent growth is a feature of oncogenic transformed cells. Libertini and colleagues optimized and validated two in vitro transformation assays based on this criterion to be used on CRISPR-Cas9 genome-edited cells. Results demonstrate that these assays could be used for preclinical safety assessment of gene therapy products.

Introduction Gene therapy have recently attracted much attention as a curative therapeutic option for inherited single gene disorders such as hemophilia. Hemophilia is a hereditary bleeding disorder caused by the deficiency of clotting activity of factor VIII (FVIII) or factor IX (FIX), and gene therapy for hemophilia using viral vector have been vigorously investigated worldwide. Toward further advancement of gene therapy for hemophilia, we have previously developed and validated the efficacy of novel two types of gene transfer technologies using a mouse model of hemophilia A. Here we investigated the efficacy and safety of the technologies in canine model. Especially, validations of technical procedures of the gene transfers for dogs were focused. Methods Green fluorescence protein (GFP) gene were transduced into normal beagle dogs by ex vivo and in vivo gene transfer techniques. For ex vivo gene transfer, blood outgrowth endothelial cells (BOECs) derived from peripheral blood of normal dogs were transduced with GFP gene using lentivirus vector, propagated, fabricated as cell sheets, then implanted onto the omentum of the same dogs. For in vivo gene transfer, normal dogs were subjected to GFP gene transduction with non-viral piggyBac vector by liver-targeted hydrodynamic injections. Results No major adverse events were observed during the gene transfers in both gene transfer systems. As for ex vivo gene transfer, histological findings from the omental biopsy performed 4 weeks after implantation revealed the tube formation by implanted GFP-positive BOECs in the sub-adipose tissue layer without any inflammatory findings, and the detected GFP signals were maintained over 6 months. Regarding in vivo gene transfer, analyses of liver biopsy samples revealed more than 90% of liver cells were positive for GFP signals in the injected liver lobes 1 week after gene transfers, then the signals gradually declined overtime. Conclusions Two types of gene transfer techniques were successfully applied to a canine model, and the transduced gene expressions persisted for a long term. Toward clinical application for hemophilia patients, practical assessments of therapeutic efficacy of these techniques will need to be performed using a dog model of hemophilia and FVIII (or FIX) gene.

Adeno-associated virus (AAV) is a promising gene therapy vector, but questions remain regarding mechanisms of basic viral functions. We previously showed that a serine/threonine (S/T) triplet motif and its flanking residues, located in the overlapping N-terminus of VP1/VP2 and highly conserved across most AAV serotypes, are critical for viral transcript production in vitro. Here we generate a panel of S/T triplet mutants in AAV serotypes 2, 4, and 9 and characterize their behaviors in vitro and in vivo using next generation sequencing. We show that S/T triplet mutations can significantly hinder some stages of transduction in a serotype-dependent manner in vitro. Interestingly, these defects are largely overcome in C57BL/6 mice, with only one mutant displaying altered behavior in vivo. Taken together, our results identify a short N-terminal capsid motif with diverse roles across several AAV serotypes which better informs engineering efforts to improve AAV as a vector for gene therapy.

Detecting familial hypercholesterolemia (FH) early and “normalizing” low-density lipoprotein (LDL) cholesterol values are the 2 pillars for effective cardiovascular disease prevention in FH. Combining lipid-lowering therapies targeting synergistic/complementary metabolic pathways makes this feasible, even among severe phenotypes. For LDL receptor-dependent treatments, PCSK9 remains the main target for adjunctive therapy to statins and ezetimibe through a variety of approaches. These include protein inhibition (adnectins), inhibition of translation at mRNA level (antisense oligonucleotides or small interfering RNA), and creation of loss-of-function mutations through base-pair editing. For patients with little LDL receptor function, LDL receptor-independent treatment targeting ANGPTL3 through monoclonal therapies are now available, or in the future, antisense/small interfering RNA-based approaches offer alternative approaches. Finally, first-in-human studies are ongoing, testing adenovirus-mediated gene therapy transducing healthy LDLR DNA in patients with HoFH. Further development of the CRISPR cas technology, which has shown promising results in vivo on introducing PCSK9 loss-of-function mutations, will move a single-dose, curative treatment for FH closer.

Erectile dysfunction affects an increasing number of men. The mainstays of management include oral medications, local erectogenic agents, and surgical placement of prosthetic devices. Newer technologies such as stem cell and gene therapy have been investigated as a means to restore spontaneous erectile capacity. Mesenchymal stem cells are thought to produce a local immunomodulatory and pro-repair milieu at the area of injury or needed repair. Gene therapy involves targeting the erectogenic pathway to augment factors involved in producing a natural erection. Such therapies are considered experimental and should be used in the setting of a clinical trial with appropriate oversight.

With the development of CRISPR-Cas9-mediated gene-editing technologies, correction of disease-causing mutations has become possible. However, current gene-correction strategies preclude mutation repair in post-mitotic cells of human tissues, and a unique repair strategy must be designed and tested for each and every mutation that may occur in a gene. We have developed a novel gene-correction strategy, co-opting regulation bypass repair (CRBR), which can repair a spectrum of mutations in mitotic or post-mitotic cells and tissues. CRBR utilizes the non-homologous end joining (NHEJ) pathway to insert a coding sequence (CDS) and transcription/translation terminators targeted upstream of any CDS mutation and downstream of the transcriptional promoter. CRBR results in simultaneous co-option of the endogenous regulatory region and bypass of the genetic defect. We validated the CRBR strategy for human gene therapy by rescuing a mouse model of Wolcott-Rallison syndrome (WRS) with permanent neonatal diabetes caused by either a large deletion or a nonsense mutation in the PERK (EIF2AK3) gene. Additionally, we integrated a CRBR GFP-terminator cassette downstream of the human insulin promoter in cadaver pancreatic islets of Langerhans, which resulted in insulin promoter regulated expression of GFP, demonstrating the potential utility of CRBR in human tissue gene repair.

Objective Plant-derived cytotoxic transgene expression, such as trichosanthin (tcs), regulated by recombinant adeno-associated virus (rAAV) vector is a promising cancer gene therapy. However, the cytotoxic transgene can hamper the vector production in the rAAV producer cell line, human embryonic kidney (HEK293) cells. Here, we explored microRNA-122 (miR122) and its target sequence to limit the expression of the cytotoxic gene in the rAAV producer cells. Methods A miR122 target (122T) sequence was incorporated into the 3′ untranslated region of the tcs cDNA sequence. The firefly luciferase (fluc) transgene was used as an appropriate control. Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells. The effects of miR122 overexpression on cell growth, transgene expression, and rAAV production were determined. Results The presence of 122T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line (in vitro), fresh human hepatocytes (ex vivo), and mouse liver (in vivo). Also, the normal liver physiology was unaffected by delivery of 122T sequence by rAAV vectors. Compared with the parental cells, the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122T, as well as the ability to produce liver-targeting rAAV vectors. Fascinatingly, the yield of rAAV vectors carrying the tcs-122T gene was increased by 77.7-fold in HEK293-mir122 cells. Moreover, the tcs-122T-containing rAAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells. Conclusion HEK293-mir122 cells along with the 122T sequence provide a potential tool to attenuate the cytotoxic transgene expression, such as tcs, during rAAV vector production.

Cellular uptake of biomolecules is crucial for regulating cell function. However, powerful and biocompatible tools for dynamically manipulating the cell entry of single-stranded DNAs (ssDNAs) remain elusive. Herein, we constructed synthetic DNA circuits on the cell membrane to program the cell entry of ssDNAs, using toehold-mediated DNA strand displacement reactions. We found that the dimerization and trimerization of cholesterol-ssDNAs enhanced membrane-anchoring and cellular uptake of ssDNAs. Moreover, we demonstrated that de-dimerization and de-trimerization of cholesterol-ssDNAs could be accomplished by inputting recovery ssDNAs into the synthetic DNA circuits, which could simultaneously decrease the cellular uptake of ssDNAs. We speculate that operating the synthetic DNA circuits on the cell membrane will be a powerful strategy for regulating the cellular uptake of exogenous materials, which has important implications for bioimaging, drug delivery, and gene therapy.

After decades of setbacks, gene therapy (GT) is experiencing major breakthroughs. Five GTs have received US regulatory approval since 2017, and over 900 others are currently in development. Many of these GTs target rare pediatric diseases that are severely life-limiting, given a lack of effective treatments. As these GTs enter early-phase clinical trials, specific ethical challenges remain unresolved in three domains: evaluating risks and potential benefits, selecting participants fairly, and engaging with patient communities. Drawing on our experience as clinical investigators, basic scientists, and bioethicists involved in a first-in-human GT trial for an ultrarare pediatric disease, we analyze these ethical challenges and offer points to consider for future GT trials.

Depending upon multiple factors, malignant solid tumors are conventionally treated by some combination of surgical resection, radiation, chemotherapy, and immunotherapy. Despite decades of research, therapeutic responses remain poor for many cancer indications. Further, many current therapies in our armamentarium are either invasive or accompanied by toxic side effects. In lieu of traditional pharmaceutics and invasive therapeutic interventions, gene therapies offer more flexible and potentially more durable approaches for new anti-cancer therapies. Nonetheless, many current gene delivery approaches suffer from low transfection efficiency due to physiological barriers limiting extravasation and uptake of genetic material. Additionally, systemically administered gene therapies may lack target-specificity, which can lead to off-target effects. To overcome these challenges, many preclinical studies have shown the utility of focused ultrasound (FUS) to increase macromolecule uptake in cells and tissue under image guidance, demonstrating promise for improved delivery of therapeutics to solid tumors. As FUS-based drug delivery is now being tested in several clinical trials around the world, FUS-targeted gene therapy for solid tumor therapy may not be far behind. In this review, we comprehensively cover the literature pertaining to preclinical attempts to more efficiently deliver therapeutic genetic material with FUS and offer perspectives for future studies and clinical translation.

Recombinant adeno-associated viral (rAAV) vectors are considered promising tools for gene therapy directed at the liver. Whereas rAAV is thought to be an episomal vector, its single-stranded DNA genome is prone to intra- and inter-molecular recombination leading to rearrangements and integration into the host cell genome. Here, we ascertained the integration frequency of rAAV in human hepatocytes transduced either ex vivo or in vivo and subsequently expanded in a mouse model of xenogeneic liver regeneration. Chromosomal rAAV integration events and vector integrity were determined using the capture-PacBio sequencing approach, a long-read next-generation sequencing method that has not previously been used for this purpose. Chromosomal integrations were found at a surprisingly high frequency of 1%-3% both in vitro and in vivo. Importantly, most of the inserted rAAV sequences were heavily rearranged and were accompanied by deletions of the host genomic sequence at the integration site.

The buffering component selection is a key criterion for the formulation development process for biopharmaceuticals. This decision for recombinant adeno-associated virus (rAAV) mediated gene therapies is receiving special attention due to their rise in clinical trials which may require high concentration, frozen supply chain, and direct delivery to eye and central nervous system related sites. In the present study, we investigate the impact of rates of freezing and thawing on rAAV2 as a model serotype. It was observed that slow rate of thawing impacts rAAV2 colloidal stability in Phosphate based buffering system. Our pre-formulation workflow suggests that rAAV2 has maximum aggregation propensity between pH of 5.5 to 6.5. Thus, the overlap of maximum aggregation propensity pH range with acidic pH shift in Phosphate based buffering system during freezing and thawing appears to be responsible for 42–75% concentration drop noticed for rAAV2. This impact appears to be fully mitigated upon replacement of Phosphate based buffering system with an alternate buffer system such as Tris. The results reported in this study highlight associated risks and provide preliminary guidance on handling of early stage frozen rAAV mediated gene therapies.

Reprogramming non-cardiomyocytes (non-CMs) into cardiomyocyte (CM)-like cells is a promising strategy for cardiac regeneration in conditions such as ischemic heart disease. Here, we used a modified mRNA (modRNA) gene delivery platform to deliver a cocktail, termed 7G-modRNA, of four cardiac-reprogramming genes-Gata4 (G), Mef2c (M), Tbx5 (T), and Hand2 (H)-together with three reprogramming-helper genes-dominant-negative (DN)-TGFβ, DN-Wnt8a, and acid ceramidase (AC)-to induce CM-like cells. We showed that 7G-modRNA reprogrammed 57% of CM-like cells in vitro. Through a lineage-tracing model, we determined that delivering the 7G-modRNA cocktail at the time of myocardial infarction reprogrammed ∼25% of CM-like cells in the scar area and significantly improved cardiac function, scar size, long-term survival, and capillary density. Mechanistically, we determined that while 7G-modRNA cannot create de novo beating CMs in vitro or in vivo, it can significantly upregulate pro-angiogenic mesenchymal stromal cells markers and transcription factors. We also demonstrated that our 7G-modRNA cocktail leads to neovascularization in ischemic-limb injury, indicating CM-like cells importance in other organs besides the heart. modRNA is currently being used around the globe for vaccination against COVID-19, and this study proves this is a safe, highly efficient gene delivery approach with therapeutic potential to treat ischemic diseases.

A common feature of diverse brain disorders is the alteration of GABA-mediated inhibition because of aberrant, intracellular chloride homeostasis induced by changes in the expression and/or function of chloride transporters. Notably, pharmacological inhibition of the chloride importer NKCC1 is able to rescue brain-related core deficits in animal models of these pathologies and in some human clinical studies. Here, we show that reducing NKCC1 expression by RNA interference in the Ts65Dn mouse model of Down syndrome (DS) restores intracellular chloride concentration, efficacy of gamma-aminobutyric acid (GABA)-mediated inhibition, and neuronal network dynamics in vitro and ex vivo. Importantly, adeno-associated virus (AAV)-mediated, neuron-specific NKCC1 knockdown in vivo rescues cognitive deficits in diverse behavioral tasks in Ts65Dn animals. Our results highlight a mechanistic link between NKCC1 expression and behavioral abnormalities in DS mice and establish a molecular target for new therapeutic approaches, including gene therapy, to treat brain disorders characterized by neuronal chloride imbalance.

Duchenne muscular dystrophy (DMD) is a severe X-linked disease characterized by progressive muscle weakness. It is caused by a variety of DMD gene pathogenic variations (large deletions or duplications, and small mutations) which leads to the absence or to a decreased amount of dystrophin protein. The allelic Becker muscular dystrophy is characterized by later onset and milder muscle involvement, and other rarer phenotypes might also be associated, such as dilated cardiomyopathy, cognitive impairment, and other neurological signs. Following the identification of the genetic cause and the disease pathophysiology, innovative personalized therapies emerged. These can be categorized into two main groups: (1) therapies aiming at the restoration of dystrophin at the sarcolemma; (2) therapeutics dealing with secondary consequences of dystrophin deficiency. In this review we provide an overview about DMD genotype-phenotype correlation, and on main approaches to restore dystrophin as stop codon read-through, exon skipping, vector-mediated gene therapy, and genome-editing strategies, some of these are based on approved orphan drugs. Finally, we present the clinical potential of novel strategies combining therapies to correct the genetic defect and other approaches, targeting secondary downstream pathological cascade due to dystrophin deficiency.

Gene therapy for hypercholesterolemia offers the potential to sustainably ameliorate disease for life with a single dose. In this study, we demonstrate the combinatorial effects of codon and vector optimization, which significantly improve the efficacy of an adeno-associated virus (AAV) vector in the low-density lipoprotein receptor (LDLR)-deficient mouse model (Ldlr−/−, Apobec1−/− double knockout [DKO]). This study investigated vector efficacy following the combination of intervening sequence 2 (IVS2) of the human beta-globin gene and codon optimization with the previously developed gain-of-function, human LDLR triple-mutant variant (hLDLR-L318D/K809R/C818A) in the treatment of homozygous familial hypercholesterolemia (HoFH). Vector doses as low as 3 × 1011 genome copies (GC)/kg achieved a robust reduction of serum low-density lipoprotein cholesterol (LDL-C) by 98% in male LDLR-deficient mice. Less efficient LDL-C reduction was observed in female mice, which was attributable to lower gene transfer efficiency in liver. We also observed persistent and stable transgene expression for 120 days, with LDL-C levels being undetectable in male DKO mice treated with the second-generation vector. In conclusion, codon and vector optimization enhanced transgene expression and reduced serum LDL-C levels effectively at a lower dose in LDLR-deficient mice. The second-generation clinical candidate vector we have developed has the potential to achieve therapeutic effects in HoFH patients., Graphical abstract, Combining codon and vector optimization significantly improved the efficacy of an AAV vector to treat a mouse model of homozygous familial hypercholesterolemia (HoFH). This second-generation clinical candidate vector has the potential to achieve therapeutic effects in HoFH patients and showcases translatable engineering strategies to enhance gene therapy vector efficacy.

Hypophosphatasia (HPP) is an inherited skeletal disease characterized by defective bone and tooth mineralization due to a deficiency in tissue-nonspecific alkaline phosphatase (TNALP). Patients with the severe infantile form of HPP may appear normal at birth, but their prognosis is very poor. To develop a practical gene therapy for HPP, we endeavored to phenotypically correct TNALP knockout (Akp2−/−) mice through adeno-associated virus type 8 (AAV8) vector-mediated, muscle-directed, TNALP expression. Following treatment of neonatal Akp2−/− mice with a single intramuscular injection of ARU-2801 (AAV8-TNALP-D10-vector) at 1.0 × 1012 vector genomes/body, high plasma ALP levels (19.38 ± 5.02 U/mL) were detected for up to 18 months, and computed tomography analysis showed mature bone mineralization. Histochemical staining for ALP activity in the knee joint revealed ALP activity on the surface of the endosteal bone of mice. Throughout their lives, the surviving treated Akp2−/− mice exhibited normal physical activity and a healthy appearance, whereas untreated controls died within 3 weeks. No ectopic calcification or abnormal calcium metabolism was detected in the treated mice. These findings suggest that ARU-2801-mediated neonatal intramuscular gene therapy is both safe and effective, and that this strategy could be a practical option for treatment of the severe infantile form of HPP., Graphical abstract, We succeeded in treating hypophosphatasia (HPP) model mice by a single intramuscular injection of adeno-associated virus type 8 (AAV8) vector expressing tissue-nonspecific alkaline phosphatase (TNALP) without any adverse effects. This AAV8 vector-related enzyme replacement therapy is a practical and useful approach to treating HPP patients.

In light of the intriguing potential of anti-angiogenic approach in suppressing choroidal neovascularization, we attempted to elaborate synthetic gene delivery systems encapsulating anti-angiogenic plasmid DNA as alternatives of clinical antibody-based therapeutics. Herein, block copolymer of cyclic Arg-Gly-Asp-poly(ethylene glycol)-poly(lysine-thiol) [RGD-PEG-PLys(thiol)] with multifunctional components was tailored in manufacture of core-shell DNA delivery nanoparticulates. Note that the polycationic PLys segments were electrostatically complexed with anionic plasmid DNA into nanoscaled core, and the tethered biocompatible PEG segments presented as the spatial shell (minimizing non-specific reactions in biological milieu). Furthermore, the aforementioned self-assembly was introduced with redox-responsive disulfide crosslinking due to the thiol coupling. Hence, reversible stabilities, namely stable in extracellular milieu but susceptible to disassemble for liberation of the DNA payloads in intracellular reducing microenvironment, were verified to facilitate transcellular gene transportation. In addition, RGD was installed onto the surface of the proposed self-assemblies with aim of targeted accumulation and internalization into angiogenic endothelial cells given that RGD receptors were specifically overexpressed on their cytomembrane surface. The proposed anti-angiogenic DNA therapeutics were validated to exert efficient expression of anti-angiogenic proteins in endothelial cells and elicit potent inhibition of ocular neovasculature post intravitreous administration. Hence, the present study approved the potential of gene therapy in treatment of choroidal neovascularization. In light of sustainable gene expression properties of DNA therapeutics, our proposed synthetic gene delivery system inspired prosperous potentials in long-term treatment of choroidal neovascularization, which should be emphasized to develop further towards clinical translations., Graphical abstract Image, graphical abstract

Since the introduction of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), genome editing has been broadly applied in basic research and applied biotechnology, whereas translation into clinical testing has raised safety concerns. Indeed, although frequencies and locations of off-target events have been widely addressed, little is known about their potential biological consequences in large-scale long-term settings. We have developed a long-term adverse treatment effect (LATE) in vitro assay that addresses potential toxicity of designer nucleases by assessing cell transformation events. In small-scale proof-of-principle experiments we reproducibly detected low-frequency (, Graphical abstract, The authors introduce an in vitro (“LATE”) assay that facilitates the analysis of potential long-term toxicity of designer nucleases. They provide evidence that this assay is able to detect growth-promoting events that occurred as consequence of CRISPR/Cas off-target activity.

Cardiac endothelial cells (ECs) are important targets for cardiovascular gene therapy. However, the approach of stably transducing ECs in vivo using different vectors, including adeno-associated virus (AAV), remains unexamined. Regarding this unmet need, two AAV libraries from DNA shuffling and random peptide display were simultaneously screened in a transgenic mouse model. Cardiac ECs were isolated by cell sorting for salvage of EC-targeting AAV. Two AAV variants, i.e., EC71 and EC73, enriched in cardiac EC, were further characterized for their tissue tropism. Both of them demonstrated remarkably enhanced transduction of cardiac ECs and reduced infection of liver ECs in comparison to natural AAVs after intravenous injection. Significantly, persistent transgene expression was maintained in mouse cardiac ECs in vivo for at least 4 months. The EC71 vector was selected for delivery of the endothelial nitric oxide synthase (eNOS) gene into cardiac ECs in a mouse model of myocardial infarction. Enhanced eNOS activity was observed in the mouse heart and lung, which was correlated with partially improved cardiac function. Taken together, two AAV capsids were evolved with more efficient transduction in cardiovascular endothelium in vivo, but their endothelial tropism might need to be further optimized for practical application to cardiac gene therapy., Graphical abstract, Liu et al. used a transgenic mouse model for the evolution of adeno-associated virus targeting cardiac endothelial cells. The selected vectors can mediate long-term endothelial transgene expression in mice, which provides a plausible approach for engineering gene therapy vectors targeting rare cell types in vivo.

Most oncolytic virotherapy has thus far employed viruses deficient in genes essential for replication in normal cells but not in cancer cells. Intra-tumoral injection of such viruses has resulted in clinically significant anti-tumor effects on the lesions in the vicinity of the injection sites but not on distant visceral metastases. To overcome this limitation, we have developed a receptor-retargeted oncolytic herpes simplex virus employing a single-chain antibody for targeting tumor-associated antigens (RR-oHSV) and its modified version with additional mutations conferring syncytium formation (RRsyn-oHSV). We previously showed that RRsyn-oHSV exhibits preserved antigen specificity and an ∼20-fold higher tumoricidal potency in vitro relative to RR-oHSV. Here, we investigated the in vivo anti-tumor effects of RRsyn-oHSV using human cancer xenografts in immunodeficient mice. With only a single intra-tumoral injection of RRsyn-oHSV at very low doses, all treated tumors regressed completely. Furthermore, intra-venous administration of RRsyn-oHSV resulted in robust anti-tumor effects even against large tumors. We found that these potent anti-tumor effects of RRsyn-oHSV may be associated with the formation of long-lasting tumor cell syncytia not containing non-cancerous cells that appear to trigger death of the syncytia. These results strongly suggest that cancer patients with distant metastases could be effectively treated with our RRsyn-oHSV., Graphical abstract, Exceptionally strong anti-tumor potency was achieved by intra-tumoral or intra-venous administration of receptor-retargeted syncytial oncolytic herpes simplex virus (RRsyn-oHSV). The robustness of RRsyn-oHSV was associated with the formation of long-lasting tumor cell syncytia not containing non-cancerous cells that otherwise trigger death of the syncytia before their sufficient expansion.

A major barrier to adeno-associated virus (AAV) gene therapy is the inability to re-dose patients due to formation of vector-induced neutralizing antibodies (Nabs). Tolerogenic nanoparticles encapsulating rapamycin (ImmTOR) provide long-term and specific suppression of adaptive immune responses, allowing for vector re-dosing. Moreover, co-administration of hepatotropic AAV vectors and ImmTOR leads to an increase of transgene expression even after the first dose. ImmTOR and AAV Anc80 encoding the methylmalonyl-coenzyme A (CoA) mutase (MMUT) combination was tested in a mouse model of methylmalonic acidemia, a disease caused by mutations in the MMUT gene. Repeated co-administration of Anc80 and ImmTOR was well tolerated and led to nearly complete inhibition of immunoglobulin (Ig)G antibodies to the Anc80 capsid. A more profound decrease of plasma levels of the key toxic metabolite, plasma methylmalonic acid (pMMA), and disease biomarker, fibroblast growth factor 21 (FGF21), was observed after treatment with the ImmTOR and Anc80-MMUT combination. In addition, there were higher numbers of viral genomes per cell (vg/cell) and increased transgene expression when ImmTOR was co-administered with Anc80-MMUT. These effects were dose-dependent, with the higher doses of ImmTOR providing higher vg/cell and mRNA levels, and an improved biomarker response. Combining of ImmTOR and AAV can not only block the IgG response against capsid, but it also appears to potentiate transduction and enhance therapeutic transgene expression in the mouse model., Graphical abstract, Kishimoto and colleagues extend their earlier findings showing that combining ImmTOR rapamycin-encapsulated nanoparticles with hepatotropic AAV vector leads to increased transduction, transgene expression, and suppression of IgG responses and therefore enables effective therapeutic AAV vector re-dosing in a mouse model of methylmalonic acidemia, an inborn metabolic disease.

Gene therapy is the delivery of a therapeutic gene for endogenous cellular expression with the goal of rescuing a disease phenotype. It has been used to treat an increasing number of human diseases with many strategies proving safe and efficacious in clinical trials. Gene delivery may be viral or non-viral, performed in vivo or ex vivo, and relies on gene integration or transient expression; all of these techniques have been applied to the treatment of Fabry disease. Fabry disease is a genetic disorder of the α-galactosidase A gene, GLA, that causes an accumulation of glycosphingolipids in cells leading to cardiac, renal and cerebrovascular damage and eventually death. Currently, there are no curative treatments available, and the therapies that are used have significant drawbacks. These treatment concerns have led to the advent of gene therapies for Fabry disease. The first Fabry patients to receive gene therapy were treated with recombinant lentivirus targeting their hematopoietic stem/progenitor cells. Adeno-associated virus treatments have also begun. Alternatively, the field of gene-editing is a new and rapidly growing field. Gene-editing has been used to repair disease-causing mutations or insert genes into cellular DNA. These techniques have the potential to be applied to the treatment of Fabry disease provided the concerns of gene-editing technology, such as safety and efficiency, were addressed. This review focuses on the current state of gene therapy as it is being developed for Fabry disease, including progresses and challenges as well as an overview of gene-editing and how it may be applied to correct Fabry disease-causing mutations in the future.

Although adeno-associated viral (AAV) vector-mediated retinal gene therapies have demonstrated efficacy, the mechanisms underlying dose-dependent retinal inflammation remain poorly understood. Here, we present a quantitative analysis of cellular immune response to subretinal AAV gene therapy in mice using multicolor flow cytometry with a panel of key immune cell markers. A significant increase in CD45+ retinal leukocytes was detected from day 14 post-subretinal injection of an AAV8 vector (1 × 109 genome copies) encoding green fluorescent protein (GFP) driven by a ubiquitous promoter. These predominantly consisted of infiltrating peripheral leukocytes including macrophages, natural killer cells, CD4 and CD8 T cells, and natural killer T cells; no significant change in resident microglia population was detected. This cellular response was persistent at 28 days and suggestive of type 1 cell-mediated effector immunity. High levels (80%) of GFP fluorescence were found in the microglia, implicating their role in viral antigen presentation and peripheral leukocyte recruitment. When compared against AAV.GFP in paired eyes, an equivalent dose of an otherwise identical vector encoding the human therapeutic transgene Rab-escort protein 1 (REP1) elicited a significantly diminished cellular immune response (4.2-fold; p = 0.0221). However, the distribution of immune cell populations remained similar, indicating a common mechanism of AAV-induced immune activation., Graphical abstract, The mechanism of gene therapy-associated uveitis (GTAU) following AAV-mediated retinal gene therapy remains poorly understood. Chandler, Xue, and colleagues utilize multicolor flow cytometry to characterize the cellular immune response to subretinal AAV injections in mice, demonstrating substantial populations of myeloid and T cells from 14 days post-injection.

Limb girdle muscular dystrophy (LGMD) 2A/R1, caused by mutations in the CAPN3 gene and CAPN3 loss of function, is known to play a role in disease pathogenicity. In this study, AAVrh74.tMCK.CAPN3 was delivered systemically to two different age groups of CAPN3 knockout (KO) mice; each group included two treatment cohorts receiving low (1.17 × 1014 vg/kg) and high (2.35 × 1014 vg/kg) doses of the vector and untreated controls. Treatment efficacy was tested 20 weeks after gene delivery using functional (treadmill), physiological (in vivo muscle contractility assay), and histopathological outcomes. AAV.CAPN3 gene therapy resulted in significant, robust improvements in functional outcomes and muscle physiology at low and high doses in both age groups. Histological analyses of skeletal muscle showed remodeling of muscle, a switch to fatigue-resistant oxidative fibers in females, and fiber size increases in both sexes. Safety studies revealed no organ tissue abnormalities; specifically, there was no histopathological evidence of cardiotoxicity. These results show that CAPN3 gene replacement therapy improved the phenotype in the CAPN3 KO mouse model at both doses independent of age at the time of vector administration. The improvements were supported by an absence of cardiotoxicity, showing the efficacy and safety of the AAV.CAPN3 vector as a potential gene therapy for LGMDR1., Graphical abstract, The AAVrh74.tMCK.CAPN3 vector was delivered to two different age groups of the LGMD2A/R1 mouse model, CAPN3 KO mice. 20 weeks after injection, therapy resulted in improvements in functional (treadmill), physiological (in vivo muscle contractility), and histopathological outcomes without cardiotoxicity, showing the efficacy and safety of the AAV.CAPN3 vector as a potential gene therapy for LGMDR1.

Summary Activation of NOTCH signaling in human hematopoietic stem/progenitor cells (HSPCs) by treatment with an engineered Delta-like ligand (DELTA1ext-IgG [DXI]) has enabled ex vivo expansion of short-term HSPCs, but the effect on long-term repopulating hematopoietic stem cells (LTR-HSCs) remains uncertain. Here, we demonstrate that ex vivo culture of human adult HSPCs with DXI under low oxygen tension limits ER stress in LTR-HSCs and lineage-committed progenitors compared with normoxic cultures. A distinct HSC gene signature was upregulated in cells cultured with DXI in hypoxia and, after 21 days of culture, the frequency of LTR-HSCs increased 4.9-fold relative to uncultured cells and 4.2-fold compared with the normoxia + DXI group. NOTCH and hypoxia pathways intersected to maintain undifferentiated phenotypes in cultured HSPCs. Our work underscores the importance of mitigating ER stress perturbations to preserve functional LTR-HSCs in extended cultures and offers a clinically feasible platform for the expansion of human HSPCs., Graphical abstract, Highlights • Superior NOTCH-mediated expansion of human HSPCs in hypoxic cultures • Culture under hypoxia mitigates ER stress in human HSPCs • Hypoxia potentiates NOTCH intracellular signaling in cultured human HSPCs, Larochelle and colleagues concurrently activate NOTCH and hypoxic pathways in ex vivo cultures of human adult HSPCs to enable a clinically relevant expansion of cells with long-term repopulating potential after transplantation. They characterize the molecular intersection between the two signaling pathways and demonstrate the important role of low oxygen tension in mitigating ER stress during NOTCH-mediated expansion of human HSPCs.

Neuronal ceroid lipofuscinosis (NCL) is a family of neurodegenerative diseases caused by mutations to genes related to lysosomal function. One variant, CNL11, is caused by mutations to the gene encoding the protein progranulin, which regulates neuronal lysosomal function. Absence of progranulin causes cerebellar atrophy, seizures, dementia, and vision loss. As progranulin gene therapies targeting the brain are developed, it is advantageous to focus on the retina, as its characteristics are beneficial for gene therapy development: the retina is easily visible through direct imaging, can be assessed through quantitative methods in vivo, and requires smaller amounts of adeno-associated virus (AAV). In this study we characterize the retinal degeneration in a progranulin knockout mouse model of CLN11 and study the effects of gene replacement at different time points. Mice heterologously expressing progranulin showed a reduction in lipofuscin deposits and microglia infiltration. While mice that receive systemic AAV92YF-scCAG-PGRN at post-natal day 3 or 4 show a reduction in retina thinning, mice injected intravitreally at months 1 and 6 with AAV2.7m8-scCAG-PGRN exhibit no improvement, and mice injected at 12 months of age have thinner retinas than do their controls. Thus, delivery of progranulin proves to be time sensitive and dependent on route of administration, requiring early delivery for optimal therapeutic benefit., Graphical abstract, Biallelic mutations to progranulin cause a neurodegenerative disease, CLN11. In this study, mice modeling CLN11 received intravitreal or intravenous injections of AAV carrying the transgene for progranulin at different time points. All mice transducing progranulin show reduction in lipofuscinosis, but older mice show retinal thinning while neonatal mice show improvement in retinal thickness.