A novel dual-plasmid platform provides scalable transfection yielding improved productivity and packaging across multiple AAV serotypes and genomes.

Transient transfection of mammalian cells using plasmid DNA is a standard method to produce adeno-associated virus (AAV) vectors allowing for flexible and scalable manufacture. Typically, three plasmids are used to encode the necessary components to facilitate vector production; however, a dual-plasmid system, termed pDG, was introduced over 2 decades ago demonstrating two components could be combined resulting in comparable productivity to triple transfection. We have developed a novel dual-plasmid system, pOXB, with an alternative arrangement of sequences that results in significantly increased AAV vector productivity and percentage of full capsids packaged in comparison to the pDG dual design and triple transfection. Here, we demonstrate the reproducibility of these findings across seven recombinant AAV genomes and multiple capsid serotypes as well as the scalability of the pOXB dual-plasmid transfection at 50-L bioreactor scale. Purified drug substance showed a consistent product quality profile in line with triple-transfected vectors, except for a substantial improvement in intact genomes packaged using the pOXB dual- transfection system. Furthermore, pOXB dual- and triple-transfection-based vectors performed consistently in vivo. The pOXB dual plasmid represents an innovation in AAV manufacturing resulting in significant process gains while maintaining the flexibility of a transient transfection platform.

Single Systemic Administration of a Gene Therapy Leading to Disease Treatment in Metachromatic Leukodystrophy Arsa Knock-Out Mice.

Metachromatic leukodystrophy (MLD) is a rare, inherited, demyelinating lysosomal storage disorder caused by mutations in the arylsulfatase-A gene (ARSA). In patients, levels of functional ARSA enzyme are diminished and lead to deleterious accumulation of sulfatides. Herein, we demonstrate that intravenous administration of HSC15/ARSA restored the endogenous murine biodistribution of the corresponding enzyme, and overexpression of ARSA corrected disease biomarkers and ameliorated motor deficits in Arsa KO mice of either sex. In treated Arsa KO mice, when compared with intravenously administered AAV9/ARSA, significant increases in brain ARSA activity, transcript levels, and vector genomes were observed with HSC15/ARSA Durability of transgene expression was established in neonate and adult mice out to 12 and 52 weeks, respectively. Levels and correlation between changes in biomarkers and ARSA activity required to achieve functional motor benefit was also defined. Finally, we demonstrated blood-nerve, blood-spinal and blood-brain barrier crossing as well as the presence of circulating ARSA enzyme activity in the serum of healthy nonhuman primates of either sex. Together, these findings support the use of intravenous delivery of HSC15/ARSA-mediated gene therapy for the treatment of MLD.SIGNIFICANCE STATEMENT Herein, we describe the method of gene therapy adeno-associated virus (AAV) capsid and route of administration selection leading to an efficacious gene therapy in a mouse model of metachromatic leukodystrophy. We demonstrate the therapeutic outcome of a new naturally derived clade F AAV capsid (AAVHSC15) in a disease model and the importance of triangulating multiple end points to increase the translation into higher species via ARSA enzyme activity and biodistribution profile (with a focus on the CNS) with that of a key clinically relevant biomarker.

Folleto de juegos predeportivos correctivos compensatorios para educandos con discapacidad intelectual / Folheto de jogos compensatórios pré-esportivos compensatórios para alunos com deficiência intelectual / Booklet of compensatory corrective pre-sports games for students with intellectual disabilities

RESUMEN Introducción: Los juegos predeportivos son una forma lúdica motora de tipo intermedio entre el juego y el deporte, imbrica elementos de la modalidad deportiva, encargado de revalorizar lo lúdico del deporte más allá de la existencia de un ganador o un perdedor. Objetivo: La investigación tuvo como objetivo elaborar un folleto de juegos predeportivos correctivos compensatorios para educandos con discapacidad intelectual. Materiales y métodos: Folleto que en su interior incluye un total de 15 juegos, estructurados de la siguiente forma: objetivo, nombre del juego, habilidades motrices a formar, habilidades comunicativas a formar, capacidades físicas que favorece, método a utilizar, procedimientos y formas organizativas, además cumple con los pasos metodológicos de los juegos, materiales necesarios, organización, desarrollo, reglas y variantes. Se utilizaron métodos nvestigativos tales como análisis de documentos, entrevista, encuesta, observación a clases y la triangulación metodológica. Resultados: El folleto de juegos predeportivos correctivos compensatorios constituye una más de las adecuaciones curriculares diseñadas para el proceso de enseñanza-aprendizaje en los educandos con necesidades educativas especiales, en el cual se potencia el tratamiento personalizado en correspondencia con las características psicopedagógicas que poseen los educandos. Conclusiones: El preexperimento realizado al folleto de juegos predeportivos correctivos compensatorios para educandos con discapacidad intelectual en el municipio de Pinar del Rio permitió examinar su influencia elocuente en la preparación de los educandos para una vida adulta e independiente que se reduce en mejor calidad de vida.

RESUMO Introdução: Os jogos pré-esportivos são uma forma de jogos motorizados de tipo intermediário entre jogos e esporte, elementos imbricados da modalidade esportiva, encarregados de revalorizar a ludicidade do esporte além da existência de um vencedor ou um perdedor. Objetivo: O objetivo da pesquisa era elaborar um livreto de jogos pré-esportivos compensatórios corretivos para alunos com deficiência intelectual. Materiais e métodos: O livreto inclui um total de 15 jogos, estruturados da seguinte forma: objetivo, nome do jogo, habilidades motoras a serem treinadas, habilidades comunicativas a serem treinadas, habilidades físicas a serem favorecidas, método a ser usado, procedimentos e formas organizacionais, assim como as etapas metodológicas dos jogos, materiais necessários, organização, desenvolvimento, regras e variantes. Foram utilizados métodos de pesquisa como análise de documentos, entrevista, pesquisa, observação em sala de aula e triangulação metodológica. Resultados: O livreto de jogos compensatórios corretivos pré-esportivos constitui mais uma das adaptações curriculares destinadas ao processo ensino-aprendizagem em alunos com necessidades educativas especiais, em que o tratamento personalizado é reforçado em correspondência com as características psicopedagógicas dos alunos. Conclusões: A pré-experimentação realizada no caderno de jogos pré-esportivos compensatórios corretivos para alunos com deficiência intelectual no município de Pinar del Rio permitiu examinar sua influência eloqüente na preparação dos alunos para uma vida adulta e independente que resulta em uma melhor qualidade de vida.

ABSTRACT Introduction: Pre-sports games are a form of motor play of an intermediate type between game and sport, overlapping elements of the sports modality, responsible for revaluing the playfulness of sport beyond the existence of a winner or a loser. Objective: The research aimed to develop a booklet of compensatory corrective pre-sports games for students with intellectual disabilities. Materials and methods: Booklet that includes a total of 15 games inside, structured as follows: objective, name of the game, motor skills to train, communication skills to train, physical capacities that favors, method to use, procedures and organizational forms, it also complies with the methodological steps of the games, necessary materials, organization, development, rules and variants. Research methods such as document analysis, interview, survey, class observation and methodological triangulation were used. Results: The booklet of compensatory corrective pre-sports games constitutes one more of the curricular adaptations designed for the teaching-learning process in students with special educational needs, in which the personalized treatment is enhanced in correspondence with the psychopedagogical characteristics that the students possess. Conclusions: The pre-experiment carried out on the booklet of compensatory corrective pre-sports games for students with intellectual disabilities in the municipality of Pinar del Rio allowed us to examine its eloquent influence on the preparation of students for an adult and independent life that is reduced in better quality of life.

Natural variations in AAVHSC16 significantly reduce liver tropism and maintain broad distribution to periphery and CNS.

Adeno-associated viruses derived from human hematopoietic stem cells (AAVHSCs) are naturally occurring AAVs. Fifteen AAVHSCs have demonstrated broad biodistribution while displaying differences in transduction. We examine the structure-function relationships of these natural amino acid variations on cellular binding. We demonstrate that AAVHSC16 is the only AAVHSC that does not preferentially bind to terminal galactose. AAVHSC16 contains two unique amino acids, 501I and 706C, compared with other AAVHSCs. Through mutagenesis, we determined that residue 501 contributes to the lack of galactose binding. Structural analysis revealed that residue 501 is in proximity to the galactose binding pocket, hence confirming its functional role in galactose binding. Biodistribution analysis of AAVHSC16 indicated significantly less liver tropism in mice and non-human primates compared with other clade F members, likely associated with overall binding differences observed in vitro. AAVHSC16 maintained robust tropism to other key tissues in the peripheral and central nervous systems after intravenous injection, including to the brain, heart, and gastrocnemius. Importantly, AAVHSC16 did not induce elevated liver enzyme levels in non-human primates after intravenous injection at high doses. The unique glycan binding and tropism of AAVHSC16 makes this naturally occurring capsid an attractive candidate for therapies requiring less liver tropism while maintaining broad biodistribution.

Expression of voltage-gated Ca2+ channels, Insp3Rs, and RyRs in the immature mouse ovary.

BACKGROUND: The postnatal mammalian ovary undergoes a series of changes to ensure the maturation of sufficient follicles to support ovulation and fecundation over the reproductive life. It is well known that intracellular [Ca2+]i signals are necessary for ovulation, fertilization, and egg activation. However, we lack detailed knowledge of the molecular identity, cellular distribution, and functional role of Ca2+ channels expressed during folliculogenesis. In the neonatal period, ovarian maturation is controlled by protein growth factors released from the oocyte and granulosa cells. Conversely, during the early infantile period, maturation becomes gonadotropin-dependent and is controlled by granulosa and theca cells. The significance of intracellular Ca2+ signaling in folliculogenesis is supported by the observation that mice lacking the expression of Ca2+/calmodulin-dependent kinase IV in granulosa cells suffer abnormal follicular development and impaired fertility. RESULTS: Using immunofluorescence in frozen ovarian sections and confocal microscopy, we assessed the expression of high-voltage activated Ca2+ channel alpha subunits and InsP3 and ryanodine receptors in the postnatal period from 3 to 16 days. During the neonatal stage, oocytes from primordial and primary follicles show high expression of various Ca2+-selective channels, with granulosa and stroma cells expressing significantly less. These channels are likely involved in supporting Ca2+-dependent secretion of peptide growth factors. In contrast, during the early and late infantile periods, Ca2+ channel expression in the oocyte diminishes, increasing significantly in the granulosa and particularly in immature theca cells surrounding secondary follicles. CONCLUSIONS: The developmental switch of Ca2+ channel expression from the oocytes to the perifollicular cells likely reflects the vanishing role of the oocytes once granulosa and theca cells take control of folliculogenesis in response to gonadotropins acting on their receptors.

Molecular characterization of precise in vivo targeted gene integration in human cells using AAVHSC15.

Targeted gene integration via precise homologous recombination (HR)-based gene editing has the potential to correct genetic diseases. AAV (adeno-associated virus) can mediate nuclease-free gene integration at a disease-causing locus. Therapeutic application of AAV gene integration requires quantitative molecular characterization of the edited sequence that overcome technical obstacles such as excess episomal vector genomes and lengthy homology arms. Here we describe a novel molecular methodology that utilizes quantitative next-generation sequencing to characterize AAV-mediated targeted insertion and detects the presence of unintended mutations. The methods described here quantify targeted insertion and query the entirety of the target locus for the presence of insertions, deletions, single nucleotide variants (SNVs) and integration of viral components such as inverted terminal repeats (ITR). Using a humanized liver murine model, we demonstrate that hematopoietic stem-cell derived AAVHSC15 mediates in vivo targeted gene integration into human chromosome 12 at the PAH (phenylalanine hydroxylase) locus at 6% frequency, with no sign of co-incident random mutations at or above a lower limit of detection of 0.5% and no ITR sequences at the integration sites. Furthermore, analysis of heterozygous variants across the targeted locus using the methods described shows a pattern of strand cross-over, supportive of an HR mechanism of gene integration with similar efficiencies across two different haplotypes. Rapid advances in the application of AAV-mediated nuclease-free target integration, or gene editing, as a new therapeutic modality requires precise understanding of the efficiency and the nature of the changes being introduced to the target genome at the molecular level. This work provides a framework to be applied to homologous recombination gene editing platforms for assessment of introduced and natural sequence variation across a target site.

Sustained Correction of a Murine Model of Phenylketonuria following a Single Intravenous Administration of AAVHSC15-PAH.

Phenylketonuria is an inborn error of metabolism caused by loss of function of the liver-expressed enzyme phenylalanine hydroxylase and is characterized by elevated systemic phenylalanine levels that are neurotoxic. Current therapies do not address the underlying genetic disease or restore the natural metabolic pathway resulting in the conversion of phenylalanine to tyrosine. A family of hepatotropic clade F adeno-associated viruses (AAVs) was isolated from human CD34+ hematopoietic stem cells (HSCs) and one (AAVHSC15) was utilized to deliver a vector to correct the phenylketonuria phenotype in Pahenu2 mice. The AAVHSC15 vector containing a codon-optimized form of the human phenylalanine hydroxylase cDNA was administered as a single intravenous dose to Pahenu2 mice maintained on a phenylalanine-containing normal chow diet. Optimization of the transgene resulted in a vector that produced a sustained reduction in serum phenylalanine and normalized tyrosine levels for the lifespan of Pahenu2 mice. Brain levels of phenylalanine and the downstream serotonin metabolite 5-hydroxyindoleacetic acid were restored. In addition, the coat color of treated mice darkened following treatment, indicating restoration of the phenylalanine metabolic pathway. Taken together, these data support the potential of an AAVHSC15-based gene therapy as an investigational therapeutic for phenylketonuria patients.

Computer-aided classification of small airways dysfunction using impulse oscillometric features: a children-focused review.

Background and objective Spirometry, which is the most commonly used technique for asthma diagnosis, is often unsuitable for small children as it requires them to follow exact instructions and perform extreme inspiration and expiration maneuvers. In contrast, impulse oscillometry (IOS) is a child-friendly technique that could serve as an alternative pulmonary function test (PFT) for asthma diagnosis and control in children as it offers several advantages over spirometry. However, the complex test results of IOS may be difficult to be understood by practitioners due to its reliance on mechanical and electrical models of the human pulmonary system. Recognizing this reality, computer-aided decision systems could help to improve the utility of IOS. The main objective of this paper is to understand the current computer-aided classification research works on this topic. Methods This paper presents a methodological review of research works related to the computer-aided classification of peripheral airway obstruction using the IOS technique, which is focused on, but not limited to, asthmatic children. Publications that focused on computer-aided classification of asthma, peripheral dysfunction and/or small airway impairment (SAI) based on impulse oscillometric features were selected for this review. Results Out of the 34 articles that were identified using the selected scientific web databases and topic-related parameters, only eight met the eligibility criteria. The most relevant results of the articles reviewed are related to the performance of the different classifiers using static features which are solely based on the first pulmonary function testing measurements (IOS and spirometry). These results included an overall classifiers' accuracy performance ranging from 42.24% to 98.61%. Conclusion There is still a great opportunity to improve the utility of IOS by developing more computer-aided robust classifiers, specifically for the asthmatic children population as the classification studies performed to date (1) are limited in number, (2) include features derived from tests that are not optimally suitable for children, (3) are solely bi-class (mostly asthma and non-asthma) and therefore fail to include different degrees of peripheral obstruction for disease prevention and control and (4) lack of validation in cases that focus on multi-class classification of the different degrees of peripheral airway obstruction.

Optimization of irradiation dose to Aedes aegypti and Ae. albopictus in a sterile insect technique program.

The sterile insect technique (SIT) may offer a means to control the transmission of mosquito borne diseases. SIT involves the release of male insects that have been sterilized by exposure to ionizing radiation. We determined the effects of different doses of radiation on the survival and reproductive capacity of local strains of Aedes aegypti and Ae. albopictus in southern Mexico. The survival of irradiated pupae was invariably greater than 90% and did not differ significantly in either sex for either species. Irradiation had no significant adverse effects on the flight ability (capacity to fly out of a test device) of male mosquitoes, which consistently exceeded 91% in Ae. aegypti and 96% in Ae. albopictus. The average number of eggs laid per female was significantly reduced in Ae. aegypti at doses of 15 and 30 Gy and no eggs were laid by females that had been exposed to 50 Gy. Similarly, in Ae. albopictus, egg production was reduced at doses of 15 and 25 Gy and was eliminated at 35 Gy. In Ae. aegypti, fertility in males was eliminated at 70 Gy and was eliminated at 30 Gy in females, whereas in Ae. albopictus, the fertility of males that mated with untreated females was almost zero (0.1%) in the 50 Gy treatment and female fertility was eliminated at 35 Gy. Irradiation treatments resulted in reduced ovary length and fewer follicles in both species. The adult median survival time of both species was reduced by irradiation in a dose-dependent manner. However, sterilizing doses of 35 Gy and 50 Gy resulted in little reduction in survival times of males of Ae. albopictus and Ae. aegypti, respectively, indicating that these doses should be suitable for future evaluations of SIT-based control of these species. The results of the present study will be applied to studies of male sexual competitiveness and to stepwise evaluations of the sterile insect technique for population suppression of these vectors in Mexico.

Nonclinical comparability studies of recombinant human arylsulfatase A addressing manufacturing process changes.

Recombinant human arylsulfatase A (rhASA) is in clinical development for the treatment of patients with metachromatic leukodystrophy (MLD). Manufacturing process changes were introduced to improve robustness and efficiency, resulting in higher levels of mannose-6-phosphate and sialic acid in post-change (process B) compared with pre-change (process A) rhASA. A nonclinical comparability program was conducted to compare process A and process B rhASA. All doses were administered intrathecally. Pharmacodynamic comparability was evaluated in immunotolerant MLD mice, using immunohistochemical staining of lysosomal-associated membrane protein-1 (LAMP-1). Pharmacokinetic comparability was assessed in juvenile cynomolgus monkeys dosed once with 6.0 mg (equivalent to 100 mg/kg of brain weight) process A or process B rhASA. Biodistribution was compared by quantitative whole-body autoradiography in rats. Potential toxicity of process B rhASA was evaluated by repeated rhASA administration at doses of 18.6 mg in juvenile cynomolgus monkeys. The specific activities for process A and process B rhASA were 89 U/mg and 106 U/mg, respectively, which were both well within the target range for the assay. Pharmacodynamic assessments showed no statistically significant differences in LAMP-1 immunohistochemical staining in the spinal cord and in most of the brain areas assessed between process A and B rhASA-dosed mice. LAMP-1 staining was reduced with both process A and B rhASA compared with vehicle, supporting its activity. Concentration-time curves in cerebrospinal fluid and serum of cynomolgus monkeys were similar with process A and B rhASA. Process A and B rhASA were similar in terms of their pharmacokinetic parameters and biodistribution data. No process B rhASA-related toxicity was detected. In conclusion, manufacturing process changes did not affect the pharmacodynamic, pharmacokinetic or safety profiles of process B rhASA relative to process A rhASA.

Evaluación endoscópica de la mucosa gástrica del canino después de la administración oral de ácido acetil-salicílico en dosis terapéuticas durante 28 días / Canine gastric mucosal endoscopic evaluation after oral acetil-salicylic acid administration at therapeutic doses during 28 days

La mucosa gástrica de 18 caninos sanos fue examinada con un gastroscopio cada 7 días, durante la administración oral de ácido acetil-salicílico (aspirina) dos veces al día por un período de 28 días. Los caninos fueron divididos en 3 grupos, cada grupo constituido por seis. El grupo A como grupo control al cual no se le administró tratamiento. El grupo B al cual se le administró aspirina a dosis de 10mg/kg. El grupo C quienes recibieron una dosis de 20mg/kg. Los signos clínicos observados fueron anorexia y hematemesis, este último estuvo presente en un solo canino. Las lesiones gástricas fueron observadas sólo en los grupos B y C, las cuales aparecieron como lesiones petequiales, de petequiales a víbices, víbices, víbices a esquimóticas y úlcera. Utilizando el modelo estadístico de Kruskall-Wallis se observó que la presencia de lesiones en los grupos experimentales B y C con respecto al grupo control se debieron a la administración de aspirina. De acuerdo al modelo estadístico U de Mann-Whitney con respecto a la presencia de lesiones entre grupos B y C, no hubo diferencia significativa, es decir, ambas dosis ocasionaron efectos similares. Con respecto a la ubicación de la lesión las mismas fueron observadas en el cardias, cuerpo, fundus, antro, píloro e incisura angularis