Therapeutic Transgene Research Articles

Intrathymic AAV delivery results in therapeutic site-specific integration at TCR loci in mice

AbstractAdeno-associated virus (AAV) vectors have been successfully exploited in gene therapy applications for the treatment of several genetic disorders. AAV is considered an episomal vector, but it has been shown to integrate within the host cell genome after the generation of double-strand DNA breaks or nicks. Although AAV integration raises some safety concerns, it can also provide therapeutic benefit; the direct intrathymic injection of an AAV harboring a therapeutic transgene results in integration in T-cell progenitors and long-term T-cell immunity. To assess the mechanisms of AAV integration, we retrieved and analyzed hundreds of AAV integration sites from lymph node-derived mature T cells and compared these with liver and brain tissue from treated mice. Notably, we found that although AAV integrations in the liver and brain were distributed across the entire mouse genome, >90% of the integrations in T cells were clustered within the T-cell receptor α, β, and γ genes. More precisely, the insertion mapped to DNA breaks created by the enzymatic activity of recombination activating genes (RAGs) during variable, diversity, and joining recombination. Our data indicate that RAG activity during T-cell receptor maturation induces a site-specific integration of AAV genomes and opens new therapeutic avenues for achieving long-term AAV-mediated gene transfer in dividing cells.

A multiplexed barcode approach to simultaneously evaluate gene delivery by adeno-associated virus capsid variants in nonhuman primates.

Adeno-associated virus (AAV) vectors are widely used to deliver therapeutic transgenes to distinct tissues, including the liver. Vectors based on naturally occurring AAV serotypes as well as vectors using engineered capsids have shown variations in tissue tropism and level of transduction between different mouse models. Moreover, results obtained in rodents frequently lack translatability into large animal studies. In light of the increasing interest in AAV vectors for human gene therapy, an increasing number of studies are being performed in nonhuman primates. To keep animal numbers to a minimum and thus optimize the process of AAV capsid selection, we developed a multiplex barcoding approach to simultaneously evaluate the in vivo vector performance for a set of serotypes and capsid-engineered AAV vectors across multiple organs. Vector biodistribution and transgene expression were assessed by quantitative PCR, quantitative reverse transcription PCR, vector DNA amplicon Illumina sequencing and vRNAseq in male and female rhesus macaques simultaneously dosed with a mixture of barcoded naturally occurring or engineered AAV vectors encoding the same transgene. As expected, our findings show animal-to-animal variation in both the biodistribution and tissue transduction pattern, which was partly influenced by each animal's distinctive serological status. This method offers a robust approach to AAV vector optimization that can be used to identify and validate AAV vectors for gene delivery to potentially any anatomical site or cell type.

Gene Therapy for Intervertebral Disc Degeneration: An Overview of Current Strategies and Applications

Intervertebral disc degeneration is a pervasive condition contributing to chronic back pain, affecting up to a third of the population, with risk further increasing with age. It is a significant driver of disability for millions of Americans and others worldwide. The standard of care today is reliant on symptomatic treatment rather than addressing the root cause of disease. Surgical interventions alter the structural integrity and biomechanics of the spine, often leading to loss of function and motion, and post-operative complications. This is the basis for innovation in novel biologic treatments, including gene therapy, which aims to reestablish the optimal balance between matrix catabolism and anabolism within pathologically degenerating disc cells. This review will cover the significant advances that have led to identification of target therapeutic genes combined with regulated expression of the therapeutic transgene and successful systems for gene delivery into cells. Recent advances in viral and non-viral vectors for gene transfer, silencing of genes by RNA interference, editing of genes by clustered regularly interspaced short palindromic repeats, and modifying mammalian target of rapamycin signaling pathwayS offer promising treatment avenues. Clinical translation of these approaches, however, will require further investigation of the pathological basis of disc degeneration in addition to systematic safety measures for the adoption of gene therapy.

Transcriptomic analysis of the innate immune response to in vitro transfection of plasmid DNA

The innate immune response to cytosolic DNA is intended to protect the host from viral infections, but it can also inhibit the delivery and expression of therapeutic transgenes in gene and cell therapies. The goal of this work was to use mRNA sequencing to identify genes that may influence transfection efficiency in four different cell types (PC-3, Jurkat, HEK-293T, and primary Tcells). The highest transfection efficiency was observed in HEK-293T cells, which upregulated only 142 genes with no known antiviral functions after transfection with lipofectamine. Lipofection upregulated 1,057 cytokine-stimulated genes (CSGs) in PC-3 cells, which exhibited a significantly lower transfection efficiency. However, when PC-3 cells were transfected in serum-containing media or electroporated, the observed transfection efficiencies were significantly higher while the expression levels of cytokines and CSGs decreased. In contrast, lipofection of Jurkat and primary Tcells only upregulated a few genes, but several of the antiviral CSGs that were absent in HEK-293T cells and upregulated in PC-3 cells were observed to be constitutively expressed in Tcells, which may explain the relatively low Lipofection efficiencies observed with Tcells (8%-21% GFP+). Indeed, overexpression of one CSG (IFI16) significantly decreased transfection efficiency in HEK-293T cells.

ET-1 STEM CELL-BASED GENE THERAPY FOR MALIGNANT GLIOMA USING GENOME-EDITED HUMAN INDUCED PLURIPOTENT STEM CELLS

Abstract Glioblastoma is the most aggressive primary brain tumor, and is characterized by diffuse infiltration into the normal brain parenchyma. New therapeutic approaches targeting invasive biological behaviour are warranted. In the present study, we show that neural stem cells (NSCs) derived from CRISRP/Cas9-edited induced pluripotent stem cells (iPSCs) have high tumor-trophic migratory capacity and stable constitutive therapeutic transgene expression, which leads to strong anti-tumor effects against glioma stem cell (GSC) models. The present study provides answers to some important research questions associated with stem cell-based gene therapy. First, the tumor-trophic migratory capacities of human iPSC-derived NSCs (iPSC-NSCs), fetal NSCs, and mesenchymal stem cells (MSCs) were quantitatively evaluated by spatiotemporal methodologies. We demonstrated that iPSC-NSCs have a higher tumor-trophic migratory capacity than MSCs in the brain. Self-repulsive action and pathotropism were important for the migration of iPSC-NSCs: ephrin ligand/receptor mediated repulsion of iPSC-NSCs and CXCL12-CXCR4 interactions between GSCs and iPSC-NSCs. Second, a prodrug converting enzyme fusion gene was selected as a therapeutic gene in human iPSCs. In general, stable constitutive transgene expression by viral vectors was difficult in human iPSCs. Furthermore, viral vectors integrate randomly into the host genome, which raises concerns about transgene silencing, insertional mutagenesis, and oncogene activation. In the present study, several common insertion sites including GAPDH, ACTB, and AAVS1, were compared. The most appropriate gene locus that achieved stable constitutive transgene expression was determined via CRISPR/Cas9-mediated genome editing. Third, we revealed the novel mechanism of action using iPSC-NSCs expressing CD-UPRT, in which ferroptosis was associated with enhanced anti-tumor immune responses. We demonstrated that the established iPSC-NSCs had strong therapeutic efficacy in GSC animal models. Finally, predictive biomarkers for the efficacy of the present treatment strategy were established. We will conduct a clinical trial of this treatment strategy. This research concept can disseminate biological, medical and engineering advances.

MicroRNA-dependent suppression of biological pacemaker activity induced by TBX18

Chemically modified mRNA (CMmRNA) with selectively altered nucleotides are used to deliver transgenes, but translation efficiency is variable. We have transfected CMmRNA encoding human T-box transcription factor 18 (CMmTBX18) into heart cells or the left ventricle of rats with atrioventricular block. TBX18 protein expression from CMmTBX18 is weak and transient, but Acriflavine, an Argonaute 2 inhibitor, boosts TBX18 levels. Small RNA sequencing identified two upregulated microRNAs (miRs) in CMmTBX18-transfected cells. Co-administration of miR-1-3p and miR-1b antagomiRs with CMmTBX18 prolongs TBX18 expression invitro and invivo and is sufficient to generate electrical stimuli capable of pacing the heart. Different suppressive miRs likewise limit the expression of VEGF-A CMmRNA. Cells therefore resist translation of CMmRNA therapeutic transgenes by upregulating suppressive miRs. Blockade of suppressive miRs enhances CMmRNA expression of genes driving biological pacing or angiogenesis. Such counterstrategies constitute an approach to boost the efficacy and efficiency of CMmRNA therapies.

Self-cleaving guide RNAs enable pharmacological selection of precise gene editing events in vivo

Expression of guide RNAs in the CRISPR/Cas9 system typically requires the use of RNA polymerase III promoters, which are not cell-type specific. Flanking the gRNA with self-cleaving ribozyme motifs to create a self-cleaving gRNA overcomes this limitation. Here, we use self-cleaving gRNAs to create drug-selectable gene editing events in specific hepatocyte loci. A recombinant Adeno Associated Virus vector targeting the Albumin locus with a promoterless self-cleaving gRNA to create drug resistance is linked in cis with the therapeutic transgene. Gene expression of both are dependent on homologous recombination into the target locus. In vivo drug selection for the precisely edited hepatocytes allows >30-fold expansion of gene-edited cells and results in therapeutic levels of a human Factor 9 transgene. Importantly, self-cleaving gRNA expression is also achieved after targeting weak hepatocyte genes. We conclude that self-cleaving gRNAs are a powerful system to enable cell-type specific in vivo drug resistance for therapeutic gene editing applications.

Transient expression of factor VIII and a chronic high-fat diet induces ER stress and late hepatocyte oncogenesis

Liver-directed gene therapy using adeno-associated virus (AAV) for delivery of therapeutic transgenes has posed particular challenges over the past 25 years, and there are no FDA-approved products to date. Reasons include lack of efficacy or toxicity or both, despite acceptable efficacy and toxicology results in animals, including non-human primates. The experience with AAV-mediated gene therapy for hemophilia is a case in point. The clotting factor VIII gene was cloned in 1984, but production of the full-length recombinant protein is challenging.

Sustained Factor VIII Activity Following Single Dose of Non-Viral Integrating Gene Therapy

The current first-line treatment for Hemophilia A patients is factor replacement therapy and/or bi-specific antibodies; however, these treatments require continuous infusions over the course of the patient's life, and patients continue to have breakthrough bleeds, representing a high unmet need for a non-viral gene therapy. Unlike conventional AAV gene therapies, our proprietary piggyBac® DNA delivery platform facilitates stable genomic insertion of a therapeutic transgene as a means of developing lifelong cures of hereditary genetic deficiencies such as hemophilia A. The Super piggyBac (SPB) transposase enzyme functions by excising a DNA cargo flanked by SPB recognition ITRs and inserting into TTAA genomic sites. We have developed a non-viral, nanoparticle-based delivery system to enable piggyBac-based gene therapy. This system entails two liver-tropic nanoparticles: a lipid nanoparticle encapsulating the SPB transposase formulated as mRNA, and a second nanoparticle encapsulating a plasmid comprising the human FVIII gene, promoter, and piggyBac ITRs. This non-viral strategy provides certain advantages over AAV-based approaches: increased transgene cargo capacity beyond 4.7 kb, stable integration of the FVIII transgene into the genome, the potential for re-dosing, and potentially simpler manufacturing processes, relative to AAV-based vector production. Optimization of the human FVIII transposon was conducted, specifically to elucidate the role of promoter, transgene sequence, and UTR elements in FVIII antigen levels. A panel of transposons was prepared and FVIII levels assessed following single administration in vivo to wild type mice. The transposons with the highest FVIII performance were subsequently evaluated in long-term efficacy studies. Initial proof-of-concept studies demonstrated that LNPs separately optimized for SPB mRNA or FVIII transposon DNA delivery can be co-administered intravenously to mice. We observed that transposase mRNA delivered to the liver is converted to protein rapidly and expressed for several days. Transposon DNA delivered to mouse liver results in expression of functional human FVIII protein, though at sub-therapeutic levels at the doses evaluated here. Co-administration of the transposon and transposase nanoparticles resulted in sustained FVIII antigen levels in the anticipated therapeutic range (>50% of normal) in wild type animals. We observed a more dramatic response when the dual-nanoparticle system was evaluated in neonatal (post-natal day 1) wild type mice. Mice were co-administered the SPB mRNA LNP and the human FVIII transposon nanoparticle. Control mice were co-administered the same dose of a catalytically dead SPB transposase, which is unable to mediate genomic integration. FVIII protein reached therapeutic levels in plasma and were sustained over the full study duration (5 months), whereas control animals exhibited negligible (<2%) human FVIII levels. These findings illustrate the utility of the piggyBac DNA delivery system for treating genetic disease early in life, enabled by stable integration. Thus, piggyBac platform avoids the transient efficacy typical of conventional non-integrating gene therapy approaches. To further validate this long-term durability, a six-month efficacy study was conducted in a mouse model of hemophilia A (FVIII exon 16−/−/CD4−/−, C57BL/6). This model is deficient in FVIII as well as CD4, allowing measurement of FVIII activity levels afforded the absence of an immune response to human FVIII. We observed that a single treatment yielded 30 - 150% of human FVIII activity in a dose-responsive manner, which was generally sustained over the duration of the study. Mice treated with the same dose of human FVIII transposon and the catalytically dead transposase exhibited negligible (<2%) human FVIII activity at all timepoints evaluated. In conclusion, our results demonstrate the novelty of our piggyBac® platform and approach in providing a potential lifelong functional cure for hemophilia A. By providing stable transgene expression through genomic integration, this technology has the potential to greatly improve the lives of hemophilia A patients.

Cryopreservation does not change the performance and characteristics of allogenic mesenchymal stem cells highly over-expressing a cytoplasmic therapeutic transgene for cancer treatment

BackgroundMesenchymal stem cells (MSCs) driven gene directed enzyme prodrug therapy is a promising approach to deliver therapeutic agents to target heterogenous solid tumours. To democratize such a therapy, cryopreservation along with cold chain transportation is an essential part of the logistical process and supply chain. Previously, we have successfully engineered MSCs by a non-viral DNA transfection approach for prolonged and exceptionally high expression of the fused transgene cytosine deaminase, uracil phosphoribosyl transferase and green fluorescent protein (CD::UPRT::GFP). The aim of this study was to determine the effects of cryopreservation of MSCs engineered to highly overexpress this cytoplasmic therapeutic transgene.MethodsModified MSCs were preserved in a commercially available, GMP-grade cryopreservative—CryoStor10 (CS10) for up to 11 months. Performance of frozen-modified MSCs was compared to freshly modified equivalents in vitro. Cancer killing potency was evaluated using four different cancer cell lines. Migratory potential was assessed using matrigel invasion assay and flow cytometric analysis for CXCR4 expression. Frozen-modified MSC was used to treat canine patients via intra-tumoral injections, or by intravenous infusion followed by a daily dose of 5-flucytosine (5FC).ResultsWe found that cryopreservation did not affect the transgene expression, cell viability, adhesion, phenotypic profile, and migration of gene modified canine adipose tissue derived MSCs. In the presence of 5FC, the thawed and freshly modified MSCs showed comparable cytotoxicity towards one canine and three human cancer cell lines in vitro. These cryopreserved cells were stored for about a year and then used to treat no-option-left canine patients with two different types of cancers and notably, the patients showed progression-free interval of more than 20 months, evidence of the effectiveness in treating spontaneously occurring cancers.ConclusionThis study supports the use of cryopreserved, off-the-shelf transiently transfected MSCs for cancer treatment.

Engineering Cancer Selective Virotherapies: Are the Pieces of the Puzzle Falling into Place?

Advances in gene therapy, synthetic biology, cancer genomics, and patient-derived cancer models have expanded the repertoire of strategies for targeting human cancers using viral vectors. Novel capsids, synthetic promoters, and therapeutic payloads are being developed and assessed through approaches such as rational design, pooled library screening, and directed evolution. Ultimately, the goal is to generate precision-engineered viruses that target different facets of tumor cell biology, without compromising normal tissue and organ function. In this study, we briefly review the opportunities for engineering cancer selectivity into viral vectors at both the cell extrinsic and intrinsic level. Such stringently tumor-targeted vectors can subsequently act as platforms for the delivery of potent therapeutic transgenes, including the exciting prospect of immunotherapeutic payloads. These have the potential to eradicate nontransduced cells through stimulation of systemic anticancer immune responses, thereby side-stepping the inherent challenge of achieving gene delivery to the entire cancer cell population. We discuss the importance of using advanced primary human cellular models, such as patient-derived cultures and organoids, to enable rapid screening and triage of novel candidates using disease-relevant models. We believe this combination of improved delivery and selectivity, through novel capsids and promoters, coupled with more potent choices for the combinations of immunotherapy-based payloads seems capable of finally delivering innovative new gene therapies for oncology. Many pieces of the puzzle of how to build a virus capable of targeting human cancers appear to be falling into place.

Preclinical Evaluation of Foamy Virus Vector-Mediated Gene Addition in Human Hematopoietic Stem/Progenitor Cells for Correction of Leukocyte Adhesion Deficiency Type 1.

Ex vivo gene therapy procedures targeting hematopoietic stem and progenitor cells (HSPCs) predominantly utilize lentivirus-based vectors for gene transfer. We provide the first pre-clinical evidence of the therapeutic utility of a foamy virus vector (FVV) for the genetic correction of human leukocyte adhesion deficiency type 1 (LAD-1), an inherited primary immunodeficiency resulting from mutation of the β2 integrin common chain, CD18. CD34+ HSPCs isolated from a severely affected LAD-1 patient were transduced under a current good manufacturing practice-compatible protocol with FVV harboring a therapeutic CD18 transgene. LAD-1-associated cellular chemotactic defects were ameliorated in transgene-positive, myeloid-differentiated LAD-1 cells assayed in response to a strong neutrophil chemoattractant in vitro. Xenotransplantation of vector-transduced LAD-1 HSPCs in immunodeficient (NSG) mice resulted in long-term (∼5 months) human cell engraftment within murine bone marrow. Moreover, engrafted LAD-1 myeloid cells displayed in vivo levels of transgene marking previously reported to ameliorate the LAD-1 phenotype in a large animal model of the disease. Vector insertion site analysis revealed a favorable vector integration profile with no overt evidence of genotoxicity. These results coupled with the unique biological features of wild-type foamy virus support the development of FVVs for ex vivo gene therapy of LAD-1.

Current Views on Oncolytic Adenoviruses for Cancer Therapy

Abstract: Oncolytic viruses replicate and spread in tumors at the same time, resulting in increased cytotoxicity and the reversal of tumor immune suppression. Among other viruses, recombinant adenoviruses that replicated in tumor cells were clinically tested via intratumoral or systemic administration. Although oncolytic virus replication kills tumor cells on its own, it may also activate the immune system, which can aid in tumor control. Viruses can be modified to improve their selectivity and effectiveness. Adenovirus genomes can be easily designed to incorporate various tumor-targeting pathways and therapeutic transgenes to improve antitumor properties. Poor tumor targeting, intratumoral expansion, and virocentric immune responses are all linked to low efficacy. As a result, more effective oncolytic adenoviruses that can be used alone or in combination with chemotherapy or immunotherapy are needed. Oncolytic Adenovirus (OAds) has long been considered a potential biotherapeutic agent against various cancers due to its ability to replicate cancer cells while remaining dormant in healthy cells selectively. Several preclinical studies using genetic engineering technology have increased antitumor OAds in various cancers in recent years. Systemic OAds administration is hampered by poor targeting tropism to healthy tissues, low-level ad receptors on tumor cells, and pre-existing neutralizing antibodies. Various discoveries have been made to overcome these barriers, including stem cells, nanoparticles, polymer shielding, extracellular vesicles, hydrogels, and microparticles (MPs). These carriers may improve Oncolytic viruses’ therapeutic efficacy by improving transfection, circulatory survival, cellular interactions, specific targeting, and immune response. The structure and biology of adenoviruses, the different types of OAds, and the efficacy of different carriers in the systemic administration of OAds were all examined in this review.

Ancestral library identifies conserved reprogrammable liver motif on AAV capsid

Gene therapy is emerging as a modality in 21st-century medicine. Adeno-associated viral (AAV) gene transfer is a leading technology to achieve efficient and durable expression of a therapeutic transgene. However, the structural complexity of the capsid has constrained efforts to engineer the particle toward improved clinical safety and efficacy. Here, we generate a curated library of barcoded AAVs with mutations across a variety of functionally relevant motifs. We then screen this library invitro and invivo in mice and nonhuman primates, enabling a broad, multiparametric assessment of every vector within the library. Among the results, we note a single residue that modulates liver transduction across all interrogated models while preserving transduction in heart and skeletal muscles. Moreover, we find that this mutation can be grafted into AAV9 and leads to profound liver detargeting while retaining muscle transduction-a finding potentially relevant to preventing hepatoxicities seen in clinical studies.

Transfection innovation for large-scale AAV &amp; LV: considerations for process development through commercial manufacturing

GMP-compliant nucleic acid delivery to HEK293 cells is often a critical first step in the manufacture of advanced therapies utilizing recombinant adeno-associated virus (AAV) or lentivirus (LV) to facilitate delivery of a therapeutic transgene to patients. Accordingly, the need for safe and reproducible large-scale viral vector manufacture processes has never been greater. Mirus Bio has developed a fully synthetic, innovative transfection formulation to enable higher titer AAV and LV generation and reduce the cost of therapeutic development and manufacture to bring more life-changing doses to patients sooner.&#160;Initial viral vector process development efforts tended towards adherent cell culture and transient transfection. These were for rare indications with low demand, and lower doses were needed. Now, the industry is undergoing rapid manufacturing development to support the upcoming demand for higher prevalence and/or dosage diseases. As a result, the size and yield in manufacturing have increased, as well as the need for lower costs to enable economic manufacture.

Oncolytic Adenoviruses: The Cold War against Cancer Finally Turns Hot.

Simple SummaryImmunotherapy has revolutionized cancer treatment, as demonstrated by the tremendous success of checkpoint inhibitors in different tumor types. Unfortunately, most patients, particularly patients with non-responsive “cold” tumors, do not benefit from checkpoint inhibitors. Enter “armed” oncolytic viruses, which “cooperate” with checkpoint inhibitors to improve anticancer responses. These are genetically engineered viruses that selectively infect, replicate in, and kill cancer cells but not cells from healthy tissues; in the process, oncolytic viruses express the therapeutic proteins that they are armed with or carry. This effectively turns the infected tumors “hot” and makes them suitable for treatment with checkpoint inhibitors. The most well-studied of all the oncolytic viruses are adenoviruses. These are agents of the common cold, which makes them remarkably safe for clinical use. This review article summarizes the oncolytic adenoviruses in advanced clinical trials and presents strategies to improve their anticancer activity.Oncolytic viruses, colloquially referred to as “living drugs”, amplify themselves and the therapeutic transgenes that they carry to stimulate an immune response both locally and systemically. Remarkable exceptions aside, such as the recent 14-patient trial with the PD-1 inhibitor, dostarlimab, in mismatch repair (MMR) deficient rectal cancer, where the complete response rate was 100%, checkpoint inhibitors are not cure-alls, which suggests the need for a combination partner like oncolytic viruses to prime and augment their activity. This review focuses on adenoviruses, the most clinically investigated of all the oncolytic viruses. It covers specific design features of clinical adenoviral candidates and highlights their potential both alone and in combination with checkpoint inhibitors in clinical trials to turn immunologically “cold” and unresponsive tumors into “hotter” and more responsive ones through a domino effect. Finally, a “mix-and-match” combination of therapies based on the paradigm of the cancer-immunity cycle is proposed to augment the immune responses of oncolytic adenoviruses.

Progress in Respiratory Gene Therapy

The prospect of gene therapy for inherited and acquired respiratory disease has energized the research community since the 1980s, with cystic fibrosis, as a monogenic disorder, driving early efforts to develop effective strategies. The fact that there are still no approved gene therapy products for the lung, despite many early phase clinical trials, illustrates the scale of the challenge: In the 1990s, first-generation non-viral and viral vector systems demonstrated proof-of-concept but low efficacy. Since then, there has been steady progress toward improved vectors with the capacity to overcome at least some of the formidable barriers presented by the lung. In addition, the inclusion of features such as codon optimization and promoters providing long-term expression have improved the expression characteristics of therapeutic transgenes. Early approaches were based on gene addition, where a new DNA copy of a gene is introduced to complement a genetic mutation: however, the advent of RNA-based products that can directly express a therapeutic protein or manipulate gene expression, together with the expanding range of tools for gene editing, has stimulated the development of alternative approaches. This review discusses the range of vector systems being evaluated for lung delivery; the variety of cargoes they deliver, including DNA, antisense oligonucleotides, messenger RNA (mRNA), small interfering RNA (siRNA), and peptide nucleic acids; and exemplifies progress in selected respiratory disease indications.

Development and Assessment of Herpes Simplex Virus Type 1 (HSV-1) Amplicon Vectors with Sensory Neuron-Selective Promoters.

Background: Neurogenic detrusor overactivity (NDO) is a severe pathological condition characterized by involuntary detrusor contractions leading to urine leakage. This condition is frequent after spinal cord injury (SCI). Gene therapy for NDO requires the development of vectors that express therapeutic transgenes driven by sensory neuron-specific promoters. The aim of this study was to develop and assess tools for the characterization of sensory neuron-specific promoters in dorsal root ganglia (DRG) neurons after transduction with herpes simplex virus type 1 (HSV-1)-based amplicon defective vectors. Methods: The HSV-1 vector genome encoded two independent transcription cassettes: one expressed firefly luciferase (FLuc) driven by different promoters’ candidates (rTRPV1, rASIC3, rCGRP, or hCGRP), and the other expressed a reporter gene driven by an invariable promoter. The strength and selectivity of promoters was assessed in organotypic cultures of explanted adult DRG, or sympathetic and parasympathetic ganglia from control and SCI rats. Results: The rCGRP promoter induced selective expression in the DRG of normal rats. The rTRPV-1 promoter, which did not display selective activity in control rats, induced selective expression in DRG explanted from SCI rats. Conclusions: This study provides a methodology to assess sensory neuron-specific promoters, opening new perspectives for future gene therapy for NDO.

Mammalian Sulfatases: Biochemistry, Disease Manifestation, and Therapy.

Sulfatases are enzymes that catalyze the removal of sulfate from biological substances, an essential process for the homeostasis of the body. They are commonly activated by the unusual amino acid formylglycine, which is formed from cysteine at the catalytic center, mediated by a formylglycine-generating enzyme as a post-translational modification. Sulfatases are expressed in various cellular compartments such as the lysosome, the endoplasmic reticulum, and the Golgi apparatus. The substrates of mammalian sulfatases are sulfolipids, glycosaminoglycans, and steroid hormones. These enzymes maintain neuronal function in both the central and the peripheral nervous system, chondrogenesis and cartilage in the connective tissue, detoxification from xenobiotics and pharmacological compounds in the liver, steroid hormone inactivation in the placenta, and the proper regulation of skin humidification. Human sulfatases comprise 17 genes, 10 of which are involved in congenital disorders, including lysosomal storage disorders, while the function of the remaining seven is still unclear. As for the genes responsible for pathogenesis, therapeutic strategies have been developed. Enzyme replacement therapy with recombinant enzyme agents and gene therapy with therapeutic transgenes delivered by viral vectors are administered to patients. In this review, the biochemical substrates, disease manifestation, and therapy for sulfatases are summarized.

HydrAd: A Helper-Dependent Adenovirus Targeting Multiple Immune Pathways for Cancer Immunotherapy.

Simple SummarySolid tumors are highly immunosuppressive and develop multiple inhibitory mechanisms that must be targeted simultaneously for successful cancer immunotherapy. Adenoviral vectors are promising cancer gene therapy vectors due to their inherent ability to stimulate multiple immune pathways. Adenoviruses are well characterized, and their genomes are easily manipulated, allowing for therapeutic transgene expression. Oncolytic adenoviruses are engineered to replicate specifically in malignant cells, resulting in cancer cell lysis. However, oncolytic adenoviral vectors have limited transgene capacity. Helper-dependent adenoviral vectors have been developed with the capability of expressing multiple transgenes through removal of all viral coding sequences. We have developed a helper-dependent platform for cancer immunotherapy and demonstrate expression of up to four functional transgenes. This platform allows us to target tumors with specific inhibitory pathways using our library of immunomodulatory transgenes in a mix-and-match approach for a synchronized cancer immunotherapy strategy.For decades, Adenoviruses (Ads) have been staple cancer gene therapy vectors. Ads are highly immunogenic, making them effective adjuvants. These viruses have well characterized genomes, allowing for substantial modifications including capsid chimerism and therapeutic transgene insertion. Multiple generations of Ad vectors have been generated with reduced or enhanced immunogenicity, depending on their intended purpose, and with increased transgene capacity. The latest-generation Ad vector is the Helper-dependent Ad (HDAd), in which all viral coding sequences are removed from the genome, leaving only the cis-acting ITRs and packaging sequences, providing up to 34 kb of transgene capacity. Although HDAds are replication incompetent, their innate immunogenicity remains intact. Therefore, the HDAd is an ideal cancer gene therapy vector as its infection results in anti-viral immune stimulation that can be enhanced or redirected towards the tumor via transgene expression. Co-infection of tumor cells with an oncolytic Ad and an HDAd results in tumor cell lysis and amplification of HDAd-encoded transgene expression. Here, we describe an HDAd-based cancer gene therapy expressing multiple classes of immunomodulatory molecules to simultaneously stimulate multiple axes of immune pathways: the HydrAd. Overall, the HydrAd platform represents a promising cancer immunotherapy agent against complex solid tumors.