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.

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.

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 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.

Abstract BACKGROUND The application of single cell sequencing as a novel immune monitoring platform can enhance the ability to interrogate at unprecedented depth the single-cell level dynamic phenotypic and functional attributes of immune cells in patients undergoing cancer immunotherapy treatment. We applied single-cell sequencing analysis of PBMCs in a patient with newly-diagnosed GBM enrolled on the ATTAC II clinical trial (FDA IND BB-16530) who experienced a sustained complete radiographic response to autologous CMV pp65-LAMP RNA-pulsed DC vaccines plus GM-CSF and tetanus-diphtheria booster administered during adjuvant cycles of dose-intensified TMZ. METHODS We constructed 5’ gene expression libraries and T cell receptor enriched libraries for 10x Genomics single-cell sequencing, generated from PBMCs collected prior to and during patient immunization using dendritic cells loaded with RNA encoding the CMV matrix protein pp65 conjugated with the lysosomal associated membrane protein (LAMP) sequence. RESULTS RNA-Seq revealed dynamic changes in immune cell subsets over course of first three vaccines, including increases in cytotoxic T cells and memory T cell subsets. Increased markers of T cell activation were observed during vaccination including enhanced T cell signaling pathways, proliferative signals, and cytokine production. We found that proportion of cytotoxic T cells increased from 3.42% to 11.74% in whole PBMCs after immunization. Surprisingly, we observed very high level of frequency natural killer T (NKT) cells comprising 4.75 % of this patient’s PBMCs at baseline. After three DC vaccines, the level of NKT cells in PBMC increased up to 10%. CONCLUSIONS Single cell RNA sequencing of a patient with sustained complete response to DC vaccination during cycles of dose-intensified TMZ reveals dynamic immune activation and expansion within the cytotoxic T cells and NKT compartments. These results emphasize the importance of subset specific profiling to achieve higher resolution in monitoring immune responses compared with bulk expression profiling in patients receiving immunotherapeutic treatment.

Osteoarthritis (OA) is a pathology characterized by the loss of articular cartilage. In this study, we performed a peptidomic strategy to identify endogenous peptides (neopeptides) that are released from human osteoarthritic tissue, which may serve as disease markers. With this aim, secretomes of osteoarthritic and healthy articular cartilages obtained from knee and hip were analyzed by shotgun peptidomics. This discovery step led to the identification of 1175 different peptides, corresponding to 101 proteins, as products of the physiological or pathological turnover of cartilage extracellular matrix. Then, a targeted multiple reaction monitoring-mass spectrometry method was developed to quantify the panel of best marker candidates on a larger set of samples (n = 62). Statistical analyses were performed to evaluate the significance of the observed differences and the ability of the neopeptides to classify the tissue. Eight of them were differentially abundant in the media from wounded zones of OA cartilage compared with the healthy tissue (p < 0.05). Three neopeptides belonging to Clusterin and one from Cartilage Oligomeric Matrix Protein showed a disease-dependent decrease specifically in hip OA, whereas two from Prolargin (PRELP) and one from Cartilage Intermediate Layer Protein 1 were significantly increased in samples from knee OA. The release of one peptide from PRELP showed the best metrics for tissue classification (AUC = 0.834). The present study reveals specific neopeptides that are differentially released from knee or hip human osteoarthritic cartilage compared with healthy tissue. This evidences the intervention of characteristic pathogenic pathways in OA and provides a novel panel of peptidic candidates for biomarker development.

Adoptive cellular therapy (ACT) using transfer of tumor-specific lymphocytes is a promising way to treat tumor patients. Tumor-specific T cells can lead to tumor regression in some cancer patients who do not respond to other therapies; however, the ability to track these cells after transfer has remained limited. In this study, high-throughput deep sequencing was used to track the T cells after in vivo infusion in pediatric patients with central PNETs. Bulk peripheral blood mononuclear cells (PBMCs) are stimulated ex vivo using autologous DCs loaded with total tumor RNA. We used high-throughput T cell receptor sequencing to identify and track clones and their frequency in patients T cell isolates collected prior to adoptive cellular therapy and weekly for one month and then monthly following immunotherapy treatment. cDNA was generated with addition of a common adapter at 5 end of cDNA using RACE technology. A broad range of diversity was observed with 12772 to 33709 individual clones being identified in the different samples after ex vivo expansion. The most prevalent clone represented between 0.61 and 20% of the population depending on the patient. The patients with prolonged progression-free and overall survival maintained high frequency ex vivo expanded clonotypes for many months post infusion. We observed in these ex vivo-expanded samples significantly higher expression level of markers associated with long-lived memory T cells (CD27 and CD127) compared to short overall survival patients. In most patients, the clonal hierarchies in the ACT product did not correlate with the peak clonotype hierarchys post infusion suggesting considerable restructuring of T cell repertoire once product went in vivo. CONCLUSIONS: TCR deep sequencing can be used to quantitatively track ACT-derived T cells post infusion. Monitoring of TCR repertoire dynamics during ACT may provide an early biomarker for treatment response and clinical outcomes.

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.

Patients with relapsed or progressive medulloblastoma and PNETs (reMB/PNETs) who have failed definitive radiotherapy have a dismal prognosis and no effective salvage treatment regimens. We explored whether adoptive cellular therapy (ACT) following standard myeloablative or nonmyeloablative salvage treatment regimens was feasible, safe, and of potential clinical benefit in children and young adults with reMB/PNETs. Pediatric patients and young adults (up to age 30) scheduled for surgical resection or biopsy of first reMB/PNET after having failed definitive cranial radiation therapy +/- spinal radiation were eligible for enrollment. Amplified tumor RNA-pulsed DCs were used to expand tumor-specific lymphocytes ex vivo by co-culture with autologous lymphocytes and IL-2 within our cGMP facility. Patients were enrolled onto one of two treatment arms based on disease staging and eligibility criteria: Group A received induction chemotherapy followed by high-dose chemotherapy and hematopoietic stem cell (HDC+HSC) rescue prior to ACT. Group B received salvage chemotherapy followed by non-meyloablative lymphodepletion with cyclophosphamide and fludarabine followed by ACT. 20 subjects with reMB/PNET have been enrolled on the Re-MATCH protocol. 11 subjects have received ACT, 4 are pending treatment, and 5 were screen failures. There have been no immunotherapy related dose-limiting toxicities. One subject with disseminated (Stage M4) medulloblastoma characterized by leptomeningeal disease, thoracic bony metastasis, and extensive bone marrow infiltration, demonstrated a profound and near complete radiographic and clinical response to treatment. TCR RNA sequencing of peripheral blood lymphocytes has revealed massive clonal expansion of T cells following ACT in this subject. Clinical follow-up is ongoing for primary endpoint analysis of 12-month progression free survival. Adoptive cellular therapy employing amplified total tumor RNA-pulsed DCs as a platform for expanding tumor-specific lymphocytes is feasible, safe, and potentially effective in patients with recurrent/progressive central PNETs. This study has now progressed to a multi-site investigational phase.

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 re-administered 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 (NAb) that may reduce the effectiveness of the vector, especially if re-administration 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. We describe the IND-enabling pre-clinical 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 re-administration of rAAV9-DES-hGAA injected intramuscularly into the tibialis anterior (TA) muscle using an immune modulation strategy developed for this study. In the proposed clinical study, six adult participants with Late-Onset Pompe Disease (LOPD) will be enrolled. The goal of the immune modulation strategy is to ablate B-cells prior to 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 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 approach to gene therapy studies in the future. Patients will act as their own controls. Repeated measures, at baseline and during the 3 months following each injection, will assess the safety, biochemical, and functional impact of the vector.

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.

Human Gene Therapy Clinical DevelopmentVol. 25, No. 3 Clinical ProtocolsPhase I/II Trial of Diaphragm Delivery of Recombinant Adeno-Associated Virus Acid Alpha-Glucosidase (rAAV1-CMV-GAA) Gene Vector in Patients with Pompe DiseaseBarry J. Byrne, Barbara Smith, Cathryn Mah, Lee Ann Lawson, Saleem Islam, Manuela Corti, Daniel Martin, Nicole Dobija, Maria V. Irwin, Thomas Conlon, Brian Cleaver, Nathalie Clement, and Shelley W. CollinsBarry J. ByrneSearch for more papers by this author, Barbara SmithSearch for more papers by this author, Cathryn MahSearch for more papers by this author, Lee Ann LawsonSearch for more papers by this author, Saleem IslamSearch for more papers by this author, Manuela CortiSearch for more papers by this author, Daniel MartinSearch for more papers by this author, Nicole DobijaSearch for more papers by this author, Maria V. IrwinSearch for more papers by this author, Thomas ConlonSearch for more papers by this author, Brian CleaverSearch for more papers by this author, Nathalie ClementSearch for more papers by this author, and Shelley W. CollinsSearch for more papers by this authorPublished Online:19 Sep 2014https://doi.org/10.1089/humc.2014.2514AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited byHypertrophic Cardiomyopathy versus Storage Diseases with Myocardial Involvement26 August 2023 | International Journal of Molecular Sciences, Vol. 24, No. 17Monitoring and Management of Respiratory Function in Pompe Disease: Current Perspectives1 September 2023 | Therapeutics and Clinical Risk Management, Vol. Volume 19Immune Responses and Immunosuppressive Strategies for Adeno-Associated Virus-Based Gene Therapy for Treatment of Central Nervous System Disorders: Current Knowledge and Approaches Suyash Prasad, David P. Dimmock, Benjamin Greenberg, Jagdeep S. Walia, Chanchal Sadhu, Fatemeh Tavakkoli, and Gerald S. Lipshutz14 December 2022 | Human Gene Therapy, Vol. 33, No. 23-24What’s new and what’s next for gene therapy in Pompe disease?27 April 2022 | Expert Opinion on Biological Therapy, Vol. 22, No. 9Muscle‐directed gene therapy corrects Pompe disease and uncovers species‐specific GAA immunogenicity1 December 2021 | EMBO Molecular Medicine, Vol. 14, No. 1Hepatic expression of GAA results in enhanced enzyme bioavailability in mice and non-human primates4 November 2021 | Nature Communications, Vol. 12, No. 1Current Clinical Applications of In Vivo Gene Therapy with AAVsMolecular Therapy, Vol. 29, No. 2Preclinical Research in Glycogen Storage Diseases: A Comprehensive Review of Current Animal Models17 December 2020 | International Journal of Molecular Sciences, Vol. 21, No. 24Pompe Disease: New Developments in an Old Lysosomal Storage Disorder18 September 2020 | Biomolecules, Vol. 10, No. 9Molecular Approaches for the Treatment of Pompe Disease12 November 2019 | Molecular Neurobiology, Vol. 57, No. 2Advancements in AAV-mediated Gene Therapy for Pompe DiseaseJournal of Neuromuscular Diseases, Vol. 7, No. 1Next Generation of Adeno-Associated Virus Vectors for Gene Therapy for Human Liver DiseasesGastroenterology Clinics of North America, Vol. 48, No. 2Intravenous Injection of an AAV-PHP.B Vector Encoding Human Acid α-Glucosidase Rescues Both Muscle and CNS Defects in Murine Pompe DiseaseMolecular Therapy - Methods & Clinical Development, Vol. 12Capsid Modifications for Targeting and Improving the Efficacy of AAV VectorsMolecular Therapy - Methods & Clinical Development, Vol. 12Large-Scale Clinical Manufacturing of AAV Vectors for Systemic Muscle Gene Therapy31 March 2019Clinical Gene Therapy Trials for Pompe Disease31 March 2019Can an in vivo imaging system be used to determine localization and biodistribution of AAV5-mediated gene expression following subretinal and intravitreal delivery in mice?Experimental Eye Research, Vol. 176Innovative Therapieansätze bei hereditären neuromuskulären Erkrankungen31 August 2018 | Der Nervenarzt, Vol. 89, No. 10Pompe Disease: From Basic Science to Therapy16 August 2018 | Neurotherapeutics, Vol. 15, No. 4Gene Therapy With Regulatory T Cells: A Beneficial Alliance19 March 2018 | Frontiers in Immunology, Vol. 9Safety of Intradiaphragmatic Delivery of Adeno-Associated Virus-Mediated Alpha-Glucosidase (rAAV1-CMV-hGAA) Gene Therapy in Children Affected by Pompe Disease Manuela Corti, Cristina Liberati, Barbara K. Smith, Lee Ann Lawson, Ibrahim S. Tuna, Thomas J. Conlon, Kirsten E. Coleman, Saleem Islam, Roland W. Herzog, David D. Fuller, Shelley W. Collins, and Barry J. Byrne1 December 2017 | Human Gene Therapy Clinical Development, Vol. 28, No. 4Long-term neurologic and cardiac correction by intrathecal gene therapy in Pompe disease6 September 2017 | Acta Neuropathologica Communications, Vol. 5, No. 1Rescue of Pompe disease in mice by AAV-mediated liver delivery of secretable acid α-glucosidaseScience Translational Medicine, Vol. 9, No. 418Inspiratory muscle conditioning exercise and diaphragm gene therapy in Pompe disease: Clinical evidence of respiratory plasticityExperimental Neurology, Vol. 287In vivo tissue-tropism of adeno-associated viral vectorsCurrent Opinion in Virology, Vol. 21Copackaged AAV9 Vectors Promote Simultaneous Immune Tolerance and Phenotypic Correction of Pompe Disease Phillip A. Doerfler, Adrian G. Todd, Nathalie Clément, Darin J. Falk, Sushrusha Nayak, Roland W. Herzog, and Barry J. Byrne4 November 2015 | Human Gene Therapy, Vol. 27, No. 1Targeted approaches to induce immune tolerance for Pompe disease therapyMolecular Therapy - Methods & Clinical Development, Vol. 3Manufacturing of recombinant adeno-associated viral vectors for clinical trialsMolecular Therapy - Methods & Clinical Development, Vol. 3A scalable method for the production of high-titer and high-quality adeno-associated type 9 vectors using the HSV platformMolecular Therapy - Methods & Clinical Development, Vol. 3 Volume 25Issue 3Sep 2014 InformationCopyright 2014, Mary Ann Liebert, Inc.To cite this article:Barry J. Byrne, Barbara Smith, Cathryn Mah, Lee Ann Lawson, Saleem Islam, Manuela Corti, Daniel Martin, Nicole Dobija, Maria V. Irwin, Thomas Conlon, Brian Cleaver, Nathalie Clement, and Shelley W. Collins.Phase I/II Trial of Diaphragm Delivery of Recombinant Adeno-Associated Virus Acid Alpha-Glucosidase (rAAV1-CMV-GAA) Gene Vector in Patients with Pompe Disease.Human Gene Therapy Clinical Development.Sep 2014.134-163.http://doi.org/10.1089/humc.2014.2514Published in Volume: 25 Issue 3: September 19, 2014PDF download

BACKGROUND: Adoptive cellular therapy using transfer of tumor-specific lymphocytes has emerged as a potent strategy for treatment of advanced and refractory malignancies. We have employed the use of total tumor RNA (TTRNA)-pulsed DCs in the ex vivo expansion of tumor-specific lymphocytes for use in adoptive cellular therapy targeting pediatric and adult malignant brain tumors. METHODS: Total tumor RNA was extracted and amplified from resected brain tumor specimens using previously published methods. Efforts to improve recovery, fidelity of amplification, and integrity of RNA included comparative analysis of free extraction methods, SMART TM-based cDNA synthesis, and PCR primer modifications. DC generation and T cell expansion under varying culture conditions and cytokine milieus were evaluated to improve functional yields of autologous cellular products. Validation runs of multi-site collection of tumor cells and leukapheresis products for DC and T cell generation under GLP and GMP conditions were performed using standardized operating procedures. RESULTS AND CONCLUSIONS: We have optimized high quality (RNA integrity, RIN ≥ 8) TTRNA amplification from resected brain tumor specimens using as little as 1 ng of input TTRNA through incorporation of solid-phase paramagnetic bead technology for cDNA synthesis and optimized PCR conditions. We founded that TTRNA transfection up-regulates the expression of CD80, CD83, CD86, HLA-DR in mature DCs. The feasibility of external site collection and delivery of tumor cells and leukapheresis products for tumor RNA amplification, DC generation, and T cell expansion has been validated by our laboratory. We have recently demonstrated the safety and feasibility of this adoptive cellular therapy platform in a phase I trial of pediatric patients with relapsed medulloblastoma and PNETs in a single institutional setting. A multi-institutional phase 2 clinical trial is underway (Re-MATCH trial: FDA IND BB-14058, UF IRB 128-2013).

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 to remove 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.

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.

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.

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.