Gene Transfer Studies Research Articles

Achievement of Normal Circulating Factor VIII Activity Following Bmn 270 AAV5-FVIII Gene Transfer: Interim, Long-Term Efficacy and Safety Results from a Phase 1/2 Study in Patients with Severe Hemophilia a

▪Background: As an X-linked single gene disorder of Factor VIII (FVIII), hemophilia A (HA) is an ideal candidate for gene therapy. To date, extended follow-up of multiple subjects in multiple dose cohorts following successful gene transfer in HA patients has not been reported.Aim: We present results from an ongoing Phase 1/2 study of AAV5-FVIII gene transfer in patients with severe HA receiving BMN 270. Data is reported herein from the two highest dose cohorts: 6E13 vg/kg and 4E13 vg/kg.Methods: 13 subjects (6E13 vg/kg, n=7; 4E13 vg/kg, n=6) received a single intravenous (IV) dose of an AAV5 vector containing the B-domain-deleted F8 gene (BMN 270). Safety, efficacy, immunogenicity, and other clinically relevant endpoints are being evaluated.Results: Data are current as of July 28, 2017. FVIII activity over time is presented herein as median levels over 4-week intervals. In the 6E13 vg/kg cohort, FVIII activity plateaued by 20 weeks post-BMN 270, with median levels between Weeks 20 to 64 in the normal range (86-122 IU/dL); at Week 64, median FVIII activity was 86 IU/dL (interquartile range [IQR] 65-107 IU/dL). In the 4E13 vg/kg cohort, median [IQR] FVIII activity levels continue to steadily increase toward the normal range (Week 20 [n=6]: 34 [28-40] IU/dL; Week 32 [n=3]: 51 [48-54] IU/dL). Median [IQR] annualized FVIII infusions in the 6E13 and 4E13 vg/kg cohorts declined from 139 [122-157] and 156 [112-183] to 0 [0-0] and 0 [0-0], respectively, 4 weeks post-BMN 270 infusion through the last follow-up visit. Median [IQR] annualized bleeding rate for subjects previously on prophylactic therapy (n=6 in both the 6E13 and 4E13 vg/kg cohorts) declined from 17 [1-24] and 8 [1-15] before dosing to 0 [0-0] and 0 [0-0], respectively, 4 weeks post-BMN 270 infusion through the last follow-up visit, with 5 subjects in each cohort self-reporting no bleeding episodes. Mild, grade 1, asymptomatic alanine aminotransferase (ALT) increases were reported in all 7 and 4 of 6 subjects in the 6E13 and 4E13 vg/kg cohorts, respectively. Peak ALT levels ranged from 44-141 U/L (upper limit of normal=43 U/L). All but 1 subject (in 4E13 vg/kg cohort) currently have a normal ALT level, and all but 1 subject (in 4E13 vg/kg cohort) are off of corticosteroid therapy (dose being tapered in 1 subject).Conclusions: AAV5-FVIII gene transfer in subjects with severe HA has resulted in sustained, clinically relevant FVIII activity that profoundly reduced self-reported bleeding and exogenous FVIII use >1.2 years post-gene transfer in the 6E13 vg/kg cohort. FVIII activity levels in the 4E13 vg/kg cohort continue to steadily rise, reaching the lower end of the normal range >0.6 years post-gene transfer. BMN 270 was well-tolerated. Efficacy and safety data up to 1.5- and 1-year follow-up for all subjects in the 6E13 and 4E13 cohorts, respectively, will be presented. Both doses appear to enable achievement of long-term normalization of FVIII activity in severe HA patients and complete protection against hemophilia-related bleeding, with a favorable safety profile. Optimal dosing of BMN 270 will be further evaluated in upcoming Phase 3 clinical trials. DisclosuresPasi:BioMarin Pharmaceutical Inc.; Octapharma, Alnylam Pharmaceuticals, Bioverativ: Research Funding; Pfizer, SOBI, Octapharma, Shire, Bayer, Alnylam, Biomarin, Biotest: Honoraria; Alnylam Pharmaceuticals, Inc; Biogen Idec Inc.; BioMarin Pharmaceutical Inc.; Octapharmal; Roche; Shire; SOBI: Consultancy; Alnylam Pharmaceuticals; BioMarin Pharmaceutical Inc.; SOBI: Membership on an entity's Board of Directors or advisory committees; Bayer HealthCare; Biotest; Novo Nordisk; Pfizer Inc.; Roche: Speakers Bureau; Roche, NovoNordisk, Pfizer: Other: paid instructor. Rangarajan:Pfizer: Research Funding; Shire: Research Funding; Novo Nordisk: Research Funding; BioMarin: Research Funding; Grifols: Research Funding; Alnylam: Other: Conference support; LFB: Other: Conference support. Kim:BioMarin: Employment. Lester:Sobi: Honoraria; LFB: Honoraria; Novo Nordisk: Honoraria; Bayer: Honoraria. Perry:BioMarin: Research Funding; Novo Nordisk: Honoraria; Biogen: Honoraria. Madan:BioMarin: Honoraria; Novo Nordisk: Other: Conference support; CSL Behring: Other: Conference support; Pfizer: Other: Conference support. Tavakkoli:BioMarin: Employment. Yang:BioMarin: Employment. Pierce:Roche: Consultancy; BioMarin: Consultancy. Wong:BioMarin: Employment.

Investigating Immune Responses to the scAAV9-HEXM Gene Therapy Treatment in Tay-Sachs Disease and Sandhoff Disease Mouse Models.

GM2 gangliosidosis disorders are a group of neurodegenerative diseases that result from a functional deficiency of the enzyme β-hexosaminidase A (HexA). HexA consists of an α- and β-subunit; a deficiency in either subunit results in Tay–Sachs Disease (TSD) or Sandhoff Disease (SD), respectively. Viral vector gene transfer is viewed as a potential method of treating these diseases. A recently constructed isoenzyme to HexA, called HexM, has the ability to effectively catabolize GM2 gangliosides in vivo. Previous gene transfer studies have revealed that the scAAV9-HEXM treatment can improve survival in the murine SD model. However, it is speculated that this treatment could elicit an immune response to the carrier capsid and “non-self”-expressed transgene. This study was designed to assess the immunocompetence of TSD and SD mice, and test the immune response to the scAAV9-HEXM gene transfer. HexM vector-treated mice developed a significant anti-HexM T cell response and antibody response. This study confirms that TSD and SD mouse models are immunocompetent, and that gene transfer expression can create an immune response in these mice. These mouse models could be utilized for investigating methods of mitigating immune responses to gene transfer-expressed “non-self” proteins, and potentially improve treatment efficacy.

Osteoinduction within adipose tissue fragments by heterodimeric bone morphogenetic Proteins-2/6 and -2/7 versus homodimeric bone morphogenetic protein-2: Therapeutic implications for bone regeneration.

Fragments of subcutaneous adipose tissue that have been genetically modified to express bone morphogenetic protein-2 (BMP-2) regenerate large segmental osseous lesions in rodents. Gene-activated adipose tissue can be implanted into osseous defects without prior cell extraction and cell culture. The present study aimed to explore whether the heterodimers BMP-2/6 or BMP-2/7 exceed the osteoinductive effect of BMP-2 on adipose tissue. In an in vitro tissue culture system, freshly harvested rat subcutaneous adipose tissue was cultivated in the presence of either BMP-2 or BMP-2/6 or BMP-2/7 at a high (200 ng/ml) and low (50 ng/ml) concentration. Gene expression analysis as well as histological and immunohistochemical methods were applied to test for osteoinduction. A concentration of 200 ng/ml of homodimeric BMP-2 induced osteogenic differentiation most potently, showing more calcification and a higher expression level of bone markers than both concentrations of BMP-2/6 or -2/7. A concentration of 50 ng/ml of BMP-2 was a significantly stronger osteogenic inducer than both concentrations of BMP-2/6 and the low concentration of BMP-2/7. The most potent heterodimeric driver of osteoinduction was BMP-2/7 at a high concentration, demonstrating effects similar to those of BMP-2 at a low concentration. Homodimeric BMP-2 evoked osteoinduction within adipose tissue more potently and at a lower concentration than heterodimeric BMP-2/6 or BMP-2/7. This result agrees well with the fact that it might be easier to translate adipose grafts activated by homodimeric BMP-2 clinically. Preclinical in vivo gene transfer studies are necessary to confirm the results of the present study.

HEREDITARY NEUROPATHIES & ALS: P.107 Review of ophthalmologic findings following intrathecal gene transfer for giant axonal neuropathy

Giant Axonal Neuropathy (GAN) is an ultra-rare childhood onset neurodegenerative disorder of the peripheral and central nervous system. Recessive GAN mutations cause loss of function of gigaxonin, a cytoskeletal regulatory protein, leading to progressive sensorimotor and optic neuropathy, CNS involvement and respiratory failure. We report on the ophthalmologic findings of a single site, phase I, non-randomized, open label dose escalation gene transfer study for GAN (NCT02362438), a first-in-human intrathecal (IT) AAV9 mediated gene transfer. Data from 11 patients with GAN dosed at 3 dose levels; 3.5 × 1013 (1X), 1.2 × 1014 (3.3X), and 1.8 × 1014 (5X) with scAAV9-JeT-GAN with up to 3 years of follow up is analyzed. Ophthalmic evaluations include best-corrected visual acuity, color discrimination thresholds, fundus imaging, and measurement of retinal nerve fiber later (RNFL) thickness through optical coherence tomography. Photopic electroretinography including a photopic negative response and visual evoked potentials (VEP) were also recorded. GAN natural history study data is used for comparison of visual acuity and RNFL thickness as well as correlation of RNFL thickness with validated outcome measures of motor function. Cross sectional natural history analysis shows significant correlation between RNFL thickness and the MFM32 total score. Interim analysis reveals a trend towards stabilization and slowing of the expected decline in RNFL thickness and visual acuity compared to pre-trial lead in data and natural history. VEP results were variable with no consistent trends across natural history and clinical trial cohorts. The interim analysis of ophthalmologic assessments highlights several key aspects: 1) RNFL thickness, a structural assessment of optic nerve atrophy and axonal nerve degeneration at the level of the CNS is a biomarker strongly correlated to overall disease severity. In GAN patients, the decline in RNFL precedes change in visual acuity, and changes can be detected longitudinally. 2) The trend towards stabilization of RNFL thickness post gene transfer suggests that RNFL may be an objective and responsive biomarker of visual system involvement and overall nervous system degeneration in GAN. 3) Assessment of RNFL thickness post gene transfer may be used as an exploratory marker of feasibility of CNS transduction following IT gene transfer.

HEREDITARY NEUROPATHIES & ALS: P.108 First-in-human intrathecal gene transfer study for giant axonal neuropathy: Three-year interim evaluation of safety and efficacy

GAN is a rare pediatric neurodegenerative hereditary neuropathy affecting the central and peripheral nervous system. Recessive GAN mutations cause dysfunction of gigaxonin, a cytoskeletal regulatory protein, leading to progressive sensorimotor and optic neuropathy, CNS involvement and respiratory failure with death by the 2nd to 3rd decade of life. We are conducting a first-in-human intrathecal (IT) AAV9 mediated gene transfer trial for GAN (NCT02362438). This is a single site, phase I, non-randomized, open label dose escalation study.

Morphological characters and SNP markers suggest hybridization and introgression in sympatric populations of the pleurocarpous mosses Homalothecium lutescens and H. sericeum

Hybridization in bryophytes involves a fusion of gametes produced by haploid parental gametophytes of different species. The primary hybrid is thus the short-lived diploid sporophyte, which soon undergoes meiosis prior to the formation of large amounts of haploid spores. We compared morphology of gametophytes (branch leaves) and sporophytes (capsule inclination) from sympatric populations and allopatric populations of H. lutescens and H. sericeum. In addition, we used transcriptome data to select 85 nuclear SNP markers that were fixed for alternative alleles in the two species. The SNPs were used to estimate the degree of hybridization in diploid sporophytes. Our study shows that gametophytes from sympatric populations display intermediate morphology in a number of leaf characters, with exception for leaf sizes, which are markedly smaller than those in allopatric populations. None of the 100 sporophytes appeared to be primary hybrids, but 33 displayed admixing—heterozygotic expression of SNP markers or mismatch of occasional markers in homozygous condition—suggesting that extensive introgression takes place in the sympatric populations. Most sporophytes with intermediate capsule inclination, initially classed as putative hybrids, did not display admixture of nuclear SNP markers. Sixty-seven percent of admixed sporophytes have predominantly nuclear SNPs typical for H. lutescens. Our results suggest that interspecific hybridization and bidirectional introgression are relatively common in the studied sympatric populations, giving rise to viable recombinants, but not complete mixing of the parental genomes. Our study is one of the first detailed accounts of hybridization among pleurocarpous mosses, opening for future studies of gene transfer and introgression between bryophyte lineages and its role in local adaptation and long-term evolutionary diversification.

The Mouse Levator Auris Longus Muscle: An Amenable Model System to Study the Role of Postsynaptic Proteins to the Maintenance and Regeneration of the Neuromuscular Synapse.

The neuromuscular junction (NMJ) is the peripheral synapse that controls the coordinated movement of many organisms. The NMJ is also an archetypical model to study synaptic morphology and function. As the NMJ is the primary target of neuromuscular diseases and traumatic injuries, the establishment of suitable models to study the contribution of specific postsynaptic muscle-derived proteins on NMJ maintenance and regeneration is a permanent need. Considering the unique experimental advantages of the levator auris longus (LAL) muscle, here we present a method allowing for efficient electroporation-mediated gene transfer and subsequent detailed studies of the morphology and function of the NMJ and muscle fibers. Also, we have standardized efficient facial nerve injury protocols to analyze LAL muscle NMJ degeneration and regeneration. Our results show that the expression of a control fluorescent protein does not alter either the muscle structural organization, the apposition of the pre- and post-synaptic domains, or the functional neurotransmission parameters of the LAL muscle NMJs; in turn, the overexpression of MuSK, a major regulator of postsynaptic assembly, induces the formation of ectopic acetylcholine receptor clusters. Our NMJ denervation experiments showed complete reinnervation of LAL muscle NMJs four weeks after facial nerve injury. Together, these experimental strategies in the LAL muscle constitute effective methods to combine protein expression with accurate analyses at the levels of structure, function, and regeneration of the NMJ.

Review: Some Molecular Breeding Issues at Genome Level in Fruit Species

The traditional plant breeding studies preferred for centuries are still widely used. Human beings started their first selection studies within the scope of plant breeding by making use of wild plant products in order to meet their nutritional needs. Later, the first growers have discovered foreign pollination to increase the yield of some agricultural crops. Thus, they could make improvements in the vegetative characteristics of new offspring individuals by hybridization by artificial pollination in different parent plants. With a better understanding of the science of genetics, crossbreeding studies were also important in order to obtain new individuals with desired features. Plant breeding studies haave improved further in the 20th century, and the mechanisms for how to obtain new agricultural varieties with superior qualifications that will keep the desired characteristics are better understood. The rapid developments in the fields of quantitative genetics, mutation breeding, polyploidy breeding and plant tissue culture studies have formed the foundations of today's plant breeding issues. In parallel with the advances in gene cloning, direct gene transfer studies to plant tissues have also been successful. Gel, hybridization and expression systems; cell imaging by light and electron microscopy; With advances in molecular techniques such as high-density microarrays and sequencing, various areas of 'omics' such as genomics, transcriptomics, proteomics, metabalomics and phenomics have been contributed. In the 2000s, plant breeders focused more on genomic studies. Plant breeding studies are now planned with intense interest on genome-wide association studies and genome editing (genome design) studies. The aim of this study is to give brief information about some molecular breeding issues that are being carried out at the genome level in the breeding of perennial fruit species. Molecular maps in fruit breeding, selection approaches at the molecular level and the purpose of creating genetic maps based on marker-character relationship are briefly explained by presenting examples. Although there are many documents about fruit breeding issues in different titles and in very detailed contents around the world, it is important to present a summary of these issues with Turkish narration for those interested in fruit breeding in our country and to share which fruit breeding subjects are studied. Keywords: Fruit breeding, Characterization, Molecular maps, Selection, Association maps DOI: 10.7176/JSTR/6-08-08

A mouse model that is immunologically tolerant to reporter and modifier proteins

Reporter proteins have become an indispensable tool in biomedical research. However, exogenous introduction of these reporters into mice poses a risk of rejection by the immune system. Here, we describe the generation, validation and application of a multiple reporter protein tolerant ‘Tol' mouse model that constitutively expresses an assembly of shuffled reporter proteins from a single open reading frame. We demonstrate that expression of the Tol transgene results in the deletion of CD8+ T cells specific for a model epitope, and substantially improves engraftment of reporter-gene transduced T cells. The Tol strain provides a valuable mouse model for cell transfer and viral-mediated gene transfer studies, and serves as a methodological example for the generation of poly-tolerant mouse strains.

Gene therapy using a novel G6PC-S298C variant enhances the long-term efficacy for treating glycogen storage disease type Ia

The current phase I/II clinical trial for human glycogen storage disease type-Ia (GSD-Ia) (NCT 03517085) uses a recombinant adeno-associated virus (rAAV) vector expressing a codon-optimized human glucose-6-phosphatase-α (G6Pase-α or G6PC). DNA sequence changes introduced by codon-optimization can negatively impact gene expression. We therefore generated a novel variant in which a single amino acid change, S298C, is introduced into the native human G6PC sequence. Short term gene transfer study in G6pc−/− mice showed that the rAAV-G6PC-S298C vector is 3-fold more efficacious than the native rAAV-G6PC vector. We have shown previously that restoring 3% of normal hepatic G6Pase-α activity in G6pc−/− mice prevents hepatocellular adenoma/carcinoma (HCA/HCC) development and that mice harboring <3% of normal hepatic G6Pase-α activity are at risk of tumor development. We have also shown that G6Pase-α deficiency leads to hepatic autophagy impairment that can contribute to hepatocarcinogenesis. We now undertake a long-term (66-week) preclinical characterization of the rAAV-G6PC-S298C vector in GSD-Ia gene therapy. We show that the increased efficacy of rAAV-G6PC-S298C has enabled the G6pc−/− mice treated with a lower dose of this vector to survive long-term. We further show that mice expressing ≥3% of normal hepatic G6Pase-α activity do not develop hepatic tumors or autophagy impairment but mice expressing <3% of normal hepatic G6Pase-α activity display impaired hepatic autophagy with one developing HCA/HCC nodules. Our study shows that the rAAV-G6PC-S298C vector provides equal or greater efficacy to the codon optimization approach, offering a valuable alternative vector for clinical translation in human GSD-Ia.

Evaluation of Virus‐based Vectors for Cystic Fibrosis Gene Therapy

Cystic fibrosis (CF) is a life‐threatening genetic disease caused by mutations in the CF transmembrane conductance regulator gene. It is the most common genetic disease among Caucasians. There is an urgent need to create more therapies for CF since current drugs are not effective in treating all the mutations that cause this disease. Furthermore, gene therapy is a potential treatment for CF airway disease. Because of this, various viral‐based gene transfer vectors have been evaluated for their efficiency in correcting the CF airway disease phenotype. This project aimed to compare side‐by‐side the differences in the transduction efficiency and targeting of specific cell types of adenovirus (Ad)‐based and adeno‐associated virus (AAV)‐based vectors in different models of the airway. Different serotypes of AAV, namely 1, 2, 5, 6, and Ad5‐based vectors, expressing green fluorescent protein (GFP), firefly luciferase (ffLuc) or ß‐galactosidase (LacZ) were assessed in in vitro and in vivo gene transfer studies. Madin‐Darby Canine Kidney (MDCK) cells were grown submerged on plastic, and human airway epithelial (HAE) cells were grown on transwells and fed only from the basolateral side to attempt to model the human conducting airways. The viral vectors expressing GFP were tested at doses of 102–103 genome copies (GC) for AAV or plaque‐forming unit (pfu) for Ad, and the level of transduction was assessed at different time points. The data demonstrates that in MDCK and HAE cells, the highest transduction was achieved by the Ad5 vector followed by the AAV2 vector, while AAV6 had the lowest transduction. Additionally, as examined in HAE cells, the age of the culture impacts transduction efficiency. In parallel, AAV vectors 1, 2, 5, 6 (at a dose of 8×1010 – 3×1011 GC) and Ad5 vector (at a dose of 1×1011 pfu) expressing ffLuc or LacZ were intranasally delivered to mice to assess the transduction efficiency in the epithelium of both the nose and lungs. AAV6 was the most efficient at transducing the cells of the nasal epithelium of mice and AAV5, at transducing lung cells. AAV2 did not transduce airway cells in mice. In conclusion, we confirmed that the viral vectors have different transduction profiles in different models of airway epithelium. More airway models should be studied to better understand which are more useful for preclinical assessments of CF gene therapeutics.Support or Funding InformationThis project was supported by the National Heart, Lung, and Blood Institute (R25‐HL084665‐14).

Recurrent gene duplication in the angiosperm tribe Delphinieae (Ranunculaceae) inferred from intracellular gene transfer events and heteroplasmic mutations in the plastid matK gene

The study of intracellular gene transfer may allow for the detection of interesting evolutionary processes such as ancient polyploidization. We compared 24 plastid genomes (plastomes) from tribe Delphinieae, one from tribe Nigelleae and one from tribe Ranunculeae, including five newly sequenced genomes. The functional transfers of the plastids rpl32 and rps16 to the nucleus in tribe Delphinieae were identified. Unexpectedly, we discovered multiple divergent copies of the nuclear-encoded plastid rpl32 in the genus Aconitum. Phylogenetic and synonymous substitution rate analyses revealed that the nuclear-encoded plastid rpl32 underwent two major duplication events. These ancient gene duplication events probably occurred via multiple polyploidization events in Aconitum between 11.9 and 24.7 Mya. Furthermore, our sequence rate analysis indicated that the eight plastid-encoded rpl subunits in Aconitum had a significantly accelerated evolutionary rate compared to those in other genera, suggesting that highly divergent paralogs targeted to the plastid may contribute to an elevated rate of evolution in plastid rpl genes. In addition, heteroplasmy of the plastid matK from two Aconitum species suggested the existence of potentially functional plastid maturases in its plastome. Our results provide insight into the evolutionary history of the tribe Delphinieae.

Recording mobile DNA in the gut microbiota using an Escherichia coli CRISPR-Cas spacer acquisition platform

The flow of genetic material between bacteria is central to the adaptation and evolution of bacterial genomes. However, our knowledge about DNA transfer within complex microbiomes is lacking, with most studies of horizontal gene transfer (HGT) relying on bioinformatic analyses of genetic elements maintained on evolutionary timescales or experimental measurements of phenotypically trackable markers. Here, we utilize the CRISPR-Cas spacer acquisition process to detect DNA acquisition events from complex microbiota in real-time and at nucleotide resolution. In this system, an E. coli recording strain is exposed to a microbial sample and spacers are acquired from transferred plasmids and permanently stored in genomic CRISPR arrays. Sequencing and analysis of acquired spacers enables identification of the transferred plasmids. This approach allowed us to identify individual mobile elements without relying on phenotypic markers or post-transfer replication. We found that HGT into the recording strain in human clinical fecal samples can be extensive and is driven by different plasmid types, with the IncX type being the most actively transferred.

New-Generation Benzimidazole-Based Plasmid Delivery Reagents with High Transfection Efficiencies on the Mammalian Cells.

Gene transfer and gene therapy studies require high-efficiency gene delivery reagents. By transferring the piece of DNA that we are interested in, we can alter the expression of certain gene or genes to further characterize its role in the cell function or in the organism's development, metabolism, immune system, etc. Transfection reagents that enable efficient delivery of the DNA to the cells are important tools in the molecular and cellular biology studies. There are chemical products and tools that have been used for transfection of the cells but they are not as efficient as desired or they can induce cytotoxicity. It is crucial to design and generate new transfection reagents to further support the field of biotechnology, molecular studies, cellular biology, and in vitro studies relying on them. The more efficient and the less cytotoxic compounds will be especially useful for the field. We synthesized a new set of benzimidazole-based transfection reagents that have higher efficiency to carry GFP expressing plasmid in to the mammalian cells compared with the commercially available ones with low cytotoxicity. GFP expression levels were tracked by flow cytometry to determine the transfection efficiencies. Benzimidazole-based transfection reagents can be safely used for transfection studies in tissue culture as well as in gene therapy applications due to their high efficiency in the gene transfer to the mammalian cells.

NIH Guidelines April 2019.

Following the required review period initiated in August 2018, the Department of Health and Human Services, National Institutes of Health (NIH), published the final changes to the Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines) in April 2019. This amendment focused on the review, registration, and reporting requirements for human gene transfer studies. In addition, the Recombinant DNA Advisory Committee (RAC) was renamed the Novel and Exceptional Technology and Research Advisory Committee (NeXTRAC) to serve as an advisory body on emerging technologies.

Gene Transfer Study in Patients With Late Onset Pompe Disease

Gene Transfer Study in Patients With Late Onset Pompe Disease

A Gene Transfer Study for Late-Onset Pompe Disease

A Gene Transfer Study for Late-Onset Pompe Disease

Gene Transfer Study of ABO-102 in Patients With Middle and Advanced Phases of MPS IIIA Disease

Gene Transfer Study of ABO-102 in Patients With Middle and Advanced Phases of MPS IIIA Disease

Widespread gene transfer to malignant gliomas with In vitro-to-In vivo correlation

Gene therapy of malignant gliomas has shown a lack of clinical success to date due in part to inability of conventional gene vectors to achieve widespread gene transfer throughout highly disseminated tumor areas within the brain. Here, we demonstrate that newly engineered polymer-based DNA-loaded nanoparticles (DNA-NP) possessing small particle diameters (~50 nm) and non-adhesive surface polyethylene glycol (PEG) coatings efficiently penetrate brain tumor tissue as well as healthy brain parenchyma. Specifically, this brain-penetrating nanoparticle (BPN), following intracranial administration via convection enhanced delivery (CED), provides widespread transgene expression in heathy rodent striatum and an aggressive brain tumor tissue established orthotopically in rats. The ability of BPN to efficiently traverse both tissues is of great importance as the highly invasive glioma cells infiltrated into normal brain tissue are responsible for tumor recurrence. Of note, the transgene expression within the orthotopic tumor tissue occurred preferentially in glioma cells over microglial cells. We also show that three-dimensional (3D) multicellular spheroids established with malignant glioma cells, unlike conventional two-dimensional (2D) cell cultures, serve as an excellent in vitro model reliably predicting gene vector behaviors in vivo. Briefly, DNA-NP possessing greater surface PEG coverage exhibited more uniform and higher-level transgene expression both in the 3D model and in vivo, whereas the trend was opposite in 2D culture. The finding here alerts that gene transfer studies based primarily on 2D cultures should be interpreted with caution and underscores the relevance of 3D models for screening newly engineered gene vectors prior to their in vivo evaluation.

Gene transfer studies in cattle

Gene transfer studies in cattle