RNA aggregates harness the danger response for potent cancer immunotherapy.

Cancer immunotherapy remains limited by poor antigenicity and a regulatory tumor microenvironment (TME). Here, we create "onion-like" multi-lamellar RNA lipid particle aggregates (LPAs) to substantially enhance the payload packaging and immunogenicity of tumor mRNA antigens. Unlike current mRNA vaccine designs that rely on payload packaging into nanoparticle cores for Toll-like receptor engagement in immune cells, systemically administered RNA-LPAs activate RIG-I in stromal cells, eliciting massive cytokine/chemokine response and dendritic cell/lymphocyte trafficking that provokes cancer immunogenicity and mediates rejection of both early- and late-stage murine tumor models. In client-owned canines with terminal gliomas, RNA-LPAs improved survivorship and reprogrammed the TME, which became "hot" within days of a single infusion. In a first-in-human trial, RNA-LPAs elicited rapid cytokine/chemokine release, immune activation/trafficking, tissue-confirmed pseudoprogression, and glioma-specific immune responses in glioblastoma patients. These data support RNA-LPAs as a new technology that simultaneously reprograms the TME while eliciting rapid and enduring cancer immunotherapy.

Adeno-associated virus-mediated gene therapy in a patient with Canavan disease using dual routes of administration and immune modulation.

Gene replacement therapy is a rational therapeutic strategy and clinical intervention for neurodegenerative disorders like Canavan disease, a leukodystrophy caused by biallelic mutations in the aspartoacylase (ASPA) gene. We aimed to investigate whether simultaneous intravenous (i.v.) and intracerebroventricular (i.c.v.) administration of rAAV9-CB6-ASPA provides a safe and effective therapeutic strategy in an open-label, individual-patient, expanded-access trial for Canavan disease. Immunomodulation was given prophylactically prior to adeno-associated virus (AAV) treatment to prevent an immune response to ASPA or the vector capsid. The patient served as his own control, and change from baseline was assessed by clinical pathology tests, vector genomes in the blood, antibodies against ASPA and AAV capsids, levels of cerebrospinal fluid (CSF) N-acetylaspartate (NAA), brain water content and morphology, clinical status, and motor function tests. Two years post treatment, the patient's white matter myelination had increased, motor function was improved, and he remained free of typical severe epilepsy. NAA level was reduced at 3 months and remained stable up to 4 years post treatment. Immunomodulation prior to AAV exposure enables repeat dosing and has prevented an anti-transgene immune response. Dual-route administration of gene therapy may improve treatment outcomes.

mRNA aggregates harness danger response for potent cancer immunotherapy.

Messenger RNA (mRNA) has emerged as a remarkable tool for COVID-19 prevention but its use for induction of therapeutic cancer immunotherapy remains limited by poor antigenicity and a regulatory tumor microenvironment (TME). Herein, we develop a facile approach for substantially enhancing immunogenicity of tumor-derived mRNA in lipid-particle (LP) delivery systems. By using mRNA as a molecular bridge with ultrapure liposomes and foregoing helper lipids, we promote the formation of 'onion-like' multi-lamellar RNA-LP aggregates (LPA). Intravenous administration of RNA-LPAs mimics infectious emboli and elicits massive DC/T cell mobilization into lymphoid tissues provoking cancer immunogenicity and mediating rejection of both early and late-stage murine tumor models. Unlike current mRNA vaccine designs that rely on payload packaging into nanoparticle cores for toll-like receptor engagement, RNA-LPAs stimulate intracellular pathogen recognition receptors (RIG-I) and reprogram the TME thus enabling therapeutic T cell activity. RNA-LPAs were safe in acute/chronic murine GLP toxicology studies and immunologically active in client-owned canines with terminal gliomas. In an early phase first-in-human trial for patients with glioblastoma, we show that RNA-LPAs encoding for tumor-associated antigens elicit rapid induction of pro-inflammatory cytokines, mobilization/activation of monocytes and lymphocytes, and expansion of antigen-specific T cell immunity. These data support the use of RNA-LPAs as novel tools to elicit and sustain immune responses against poorly immunogenic tumors.

Pompe disease gene therapy: neural manifestations require consideration of CNS directed therapy.

Pompe disease is a neuromuscular disease caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase leading to lysosomal and cytoplasmic glycogen accumulation in neurons and striated muscle. In the decade since availability of first-generation enzyme replacement therapy (ERT) a better understanding of the clinical spectrum of disease has emerged. The most severe form of early onset disease is typically identified with symptoms in the first year of life, known as infantile-onset Pompe disease (IOPD). Infants are described at floppy babies with cardiac hypertrophy in the first few months of life. A milder form with late onset (LOPD) of symptoms is mostly free of cardiac involvement with slower rate of progression. Glycogen accumulation in the CNS and skeletal muscle is observed in both IOPD and LOPD. In both circumstances, multi-system disease (principally motoneuron and myopathy) leads to progressive weakness with associated respiratory and feeding difficulty. In IOPD the untreated natural history leads to cardiorespiratory failure and death in the first year of life. In the current era of ERT clinical outcomes are improved, yet, many patients have an incomplete response and a substantial unmet need remains. Since the neurological manifestations of the disease are not amenable to peripheral enzyme replacement, we set out to better understand the pathophysiology and potential for treatment of disease manifestations using adeno-associated virus (AAV)-mediated gene transfer, with the first clinical gene therapy studies initiated by our group in 2006. This review focuses on the preclinical studies and clinical study findings which are pertinent to the development of a comprehensive gene therapy strategy for both IOPD and LOPD. Given the advent of newborn screening, a significant focus of our recent work has been to establish the basis for repeat administration of AAV vectors to enhance neuromuscular therapeutic efficacy over the life span.

[A retrospective analysis of the management of unconscious patients from the emergency room (ER) to intensive care (ICU) at Södersjukhuset]. / Fler medvetslösa patienter bör intuberas för säkrad luftväg - Många medvetslösa vårdas också för kort tid på IVA ­ möjliga donatorer missas, visar retrospektiv analys.

Intubation and admission to ICU are vital stages in the management of unconscious patients. Treatment records for all patients who died within 5 days of admission to Södersjukhuset during 2015 were obtained. Patients with GCS <9 in the ER were selected. It was noted if the patients had been intubated, had done a CT brain scan and if they had been admitted to ICU. They were divided into one of three diagnosis groups: cardiac arrest, intracerebral hemorrhage/stroke or other. 48 of 51 cardiac arrest patients were intubated and transferred to ICU. Only 17 of 46 ICH/stroke patients were intubated, and 22 ICH/stroke patients did a CT brain scan with an unprotected airway. Possible organ donors were more difficult to detect in the cardiac arrest group (6 of 14 possible) compared with the ICH/stroke group (12 of 14 possible). Our analysis shows that improvements need to be made in the airway management of unconscious patients who have suffered an ICH or stroke, and that identification of possible organ donors amongst victims of cardiac arrest also needs to be improved.

A scalable method for the production of high-titer and high-quality adeno-associated type 9 vectors using the HSV platform.

Recombinant adeno-associated vectors based on serotype 9 (rAAV9) have demonstrated highly effective gene transfer in multiple animal models of muscular dystrophies and other neurological indications. Current limitations in vector production and purification have hampered widespread implementation of clinical candidate vectors, particularly when systemic administration is considered. In this study, we describe a complete herpes simplex virus (HSV)-based production and purification process capable of generating greater than 1 × 10(14) rAAV9 vector genomes per 10-layer CellSTACK of HEK 293 producer cells, or greater than 1 × 10(5) vector genome per cell, in a final, fully purified product. This represents a 5- to 10-fold increase over transfection-based methods. In addition, rAAV vectors produced by this method demonstrated improved biological characteristics when compared to transfection-based production, including increased infectivity as shown by higher transducing unit-to-vector genome ratios and decreased total capsid protein amounts, shown by lower empty-to-full ratios. Together, this data establishes a significant improvement in both rAAV9 yields and vector quality. Further, the method can be readily adapted to large-scale good laboratory practice (GLP) and good manufacturing practice (GMP) production of rAAV9 vectors to enable preclinical and clinical studies and provide a platform to build on toward late-phases and commercial production.

Evaluation of Readministration of a Recombinant Adeno-Associated Virus Vector Expressing Acid Alpha-Glucosidase in Pompe Disease: Preclinical to Clinical Planning.

A recombinant serotype 9 adeno-associated virus (rAAV9) vector carrying a transgene that expresses codon-optimized human acid alpha-glucosidase (hGAA, or GAA) driven by a human desmin (DES) promoter (i.e., rAAV9-DES-hGAA) has been generated as a clinical candidate vector for Pompe disease. The rAAV9-DES-hGAA vector is being developed as a treatment for both early- and late-onset Pompe disease, in which patients lack sufficient lysosomal alpha-glucosidase leading to glycogen accumulation. In young patients, the therapy may need to be readministered after a period of time to maintain therapeutic levels of GAA. Administration of AAV-based gene therapies is commonly associated with the production of neutralizing antibodies that may reduce the effectiveness of the vector, especially if readministration is required. Previous studies have demonstrated the ability of rAAV9-DES-hGAA to correct cardiac and skeletal muscle pathology in Gaa(-/-) mice, an animal model of Pompe disease. This article describes the IND-enabling preclinical studies supporting the program for a phase I/II clinical trial in adult patients with Pompe. These studies were designed to evaluate the toxicology, biodistribution, and potential for readministration of rAAV9-DES-hGAA injected intramuscularly into the tibialis anterior muscle using an immune modulation strategy developed for this study. In the proposed clinical study, six adult participants with late-onset Pompe disease will be enrolled. The goal of the immune modulation strategy is to ablate B-cells before the initial exposure of the study agent in one leg and the subsequent exposure of the same vector to the contralateral leg four months after initial dosing. The dosing of the active agent is accompanied by a control injection of excipient dosing in the contralateral leg to allow for blinding and randomization of dosing, which may also strengthen the evidence generated from gene therapy studies in the future. Patients will act as their own controls. Repeated measures, at baseline and during the three months following each dosing will assess the safety, biochemical, and functional impact of the vector.

A simplified purification protocol for recombinant adeno-associated virus vectors.

We describe a new rapid, low cost, and scalable method for purification of various recombinant adeno-associated viruses (rAAVs) from the lysates of producer cells of either mammalian or insect origin. The method takes advantage of two general biochemical properties of all characterized AAV serotypes: (i) low isoelectric point of a capsid and (ii) relative biological stability of the viral particle in the acidic environment. A simple and rapid clarification of cell lysate toremove the bulk of proteins and DNA is accomplished by utilizing inexpensive off-the-shelf reagents such as sodium citrate and citric acid. After the low-speed centrifugation step, the supernatant is subjected to cation exchange chromatography via sulfopropyl (SP) column. The eluted virus may then be further concentrated by either centrifugal spin devices or tangential flow filtration yielding material of high titer and Good Manufacturing Practice (GMP) grade biochemical purity. The protocol is validated for rAAV serotypes 2, 8, and 9. The described method makes rAAV vector technology readily available for the low budget research laboratories and could be easily adapted for a large scale GMP production format.

Preclinical potency and safety studies of an AAV2-mediated gene therapy vector for the treatment of MERTK associated retinitis pigmentosa.

Abstract Proof of concept for MERTK gene replacement therapy has been demonstrated using different viral vectors in the Royal College of Surgeon (RCS) rat, a well characterized model of recessive retinitis pigmentosa that contains a mutation in the Mertk gene. MERTK plays a key role in renewal of photoreceptor outer segments (OS) by phagocytosis of shed OS tips. Mutations in MERTK cause impaired phagocytic activity and accumulation of OS debris in the interphotoreceptor space that ultimately leads to photoreceptor cell death. In the present study, we conducted a series of preclinical potency and GLP-compliant safety evaluations of an adeno-associated virus type 2 (AAV2) vector expressing human MERTK cDNA driven by the retinal pigment epithelium-specific, VMD2 promoter. We demonstrate the potency of the vector in RCS rats by improved electroretinogram (ERG) responses in treated eyes compared with contralateral untreated controls. Toxicology and biodistribution studies were performed in Sprague-Dawley (SD) rats injected with two different doses of AAV vectors and buffer control. Delivery of vector in SD rats did not result in a change in ERG amplitudes of rod and cone responses relative to balanced salt solution control-injected eyes, indicating that administration of AAV vector did not adversely affect normal retinal function. In vivo fundoscopic analysis and postmortem retinal morphology of the vector-injected eyes were normal compared with controls. Evaluation of blood smears showed the lack of transformed cells in the treated eyes. All injected eyes and day 1 blood samples were positive for vector genomes, and all peripheral tissues were negative. Our results demonstrate the potency and safety of the AAV2-VMD2-hMERTK vector in animal models tested. A GMP vector has been manufactured and is presently in clinical trial.

Phase I/II trial of adeno-associated virus-mediated alpha-glucosidase gene therapy to the diaphragm for chronic respiratory failure in Pompe disease: initial safety and ventilatory outcomes.

Pompe disease is an inherited neuromuscular disease caused by deficiency of lysosomal acid alpha-glucosidase (GAA) leading to glycogen accumulation in muscle and motoneurons. Cardiopulmonary failure in infancy leads to early mortality, and GAA enzyme replacement therapy (ERT) results in improved survival, reduction of cardiac hypertrophy, and developmental gains. However, many children have progressive ventilatory insufficiency and need additional support. Preclinical work shows that gene transfer restores phrenic neural activity and corrects ventilatory deficits. Here we present 180-day safety and ventilatory outcomes for five ventilator-dependent children in a phase I/II clinical trial of AAV-mediated GAA gene therapy (rAAV1-hGAA) following intradiaphragmatic delivery. We assessed whether rAAV1-hGAA results in acceptable safety outcomes and detectable functional changes, using general safety measures, immunological studies, and pulmonary functional testing. All subjects required chronic, full-time mechanical ventilation because of respiratory failure that was unresponsive to both ERT and preoperative muscle-conditioning exercises. After receiving a dose of either 1×10(12) vg (n=3) or 5×10(12) vg (n=2) of rAAV1-hGAA, the subjects' unassisted tidal volume was significantly larger (median [interquartile range] 28.8% increase [15.2-35.2], p<0.05). Further, most patients tolerated appreciably longer periods of unassisted breathing (425% increase [103-851], p=0.08). Gene transfer did not improve maximal inspiratory pressure. Expected levels of circulating antibodies and no T-cell-mediated immune responses to the vector (capsids) were observed. One subject demonstrated a slight increase in anti-GAA antibody that was not considered clinically significant. These results indicate that rAAV1-hGAA was safe and may lead to modest improvements in volitional ventilatory performance measures. Evaluation of the next five patients will determine whether earlier intervention can further enhance the functional benefit.

Pompe disease gene therapy.

Pompe disease is an autosomal recessive metabolic myopathy caused by the deficiency of the lysosomal enzyme acid alpha-glucosidase and results in cellular lysosomal and cytoplasmic glycogen accumulation. A wide spectrum of disease exists from hypotonia and severe cardiac hypertrophy in the first few months of life due to severe mutations to a milder form with the onset of symptoms in adulthood. In either condition, the involvement of several systems leads to progressive weakness and disability. In early-onset severe cases, the natural history is characteristically cardiorespiratory failure and death in the first year of life. Since the advent of enzyme replacement therapy (ERT), the clinical outcomes have improved. However, it has become apparent that a new natural history is being defined in which some patients have substantial improvement following ERT, while others develop chronic disability reminiscent of the late-onset disease. In order to improve on the current clinical outcomes in Pompe patients with diminished clinical response to ERT, we sought to address the cause and potential for the treatment of disease manifestations which are not amenable to ERT. In this review, we will focus on the preclinical studies that are relevant to the development of a gene therapy strategy for Pompe disease, and have led to the first clinical trial of recombinant adeno-associated virus-mediated gene-based therapy for Pompe disease. We will cover the preliminary laboratory studies and rationale for a clinical trial, which is based on the treatment of the high rate of respiratory failure in the early-onset patients receiving ERT.