Monitoramento do horizonte tecnológico: MEDICAMENTOS para o tratamento de Doença Falciforme / Monitoring the technological horizon: MEDICINES for the treatment of sickle cell disease

CONDIÇÃO CLÍNICA: O termo doença falciforme representa um grupo de doenças hereditárias, com apresentação multissistêmica, caracterizada por hemácias de formato anormal (em formato de foice) resultante de uma alteração na estrutura da hemoglobina (Hb). As células afetadas apresentam uma maior fragilidade e são removidas da circulação e destruídas. Esse grupo de doenças se manifesta com episódios de agudização e traz repercussões que causam danos progressivos à maioria dos órgãos, incluindo cérebro, rins, pulmões, ossos e sistema cardiovascular. A anemia falciforme, um dos tipos de doença falciforme, é um dos distúrbios monogênicos graves mais comuns em todo o mundo. A anemia falciforme é causada pela presença de hemoglobina-S (HbS), ou hemoglobina falciforme, em homozigose (HbSS). A HbS ocorre quando há a substituição de uma única base nitrogenada no gene que codifica a subunidade beta da hemoglobina (ß-globina), levando a substituição de ácido glutâmico por valina na cadeia dessa proteína. Quando a HbS é desoxigenada, a troca dos aminoácidos gera interação hidrofóbica com outra molécula de hemoglobina, desencadeando uma agregação em grandes polímeros. A polimerização da HbS é o evento primário na patogênese molecular da doença falciforme, o que causa a distorção da forma da hemácia e diminuição acentuada de sua plasticidade com consequente rigidez celular. Essa rigidez prejudica a capacidade de as hemácias transitarem por pequenos vasos, o que causa vaso-oclusão, seguida de isquemia e infarto tecidual. O infarto pode ocorrer em qualquer parte do corpo e é responsável pela manifestação clínica mais precoce: a crise de dor aguda. TRATAMENTO: O gerenciamento da doença falciforme visa a melhora na perfusão tecidual, controle de dor e prevenção, e manejo de complicações associadas à anemia, crise vaso-oclusiva (CVO) e infecções. A terapia modificadora de doença com hidroxiureia é o tratamento mais eficaz para doença falciforme até o momento. O efeito primário da terapia com hidroxiureia está relacionado ao aumento da produção da hemoglobina-fetal (HbF), reduzindo os níveis de HbS e, consequentemente, a falcização de hemácias e vaso-oclusão. De acordo com uma revisão sistemática da Cochrane de estudos clínicos randomizados e quase-randomizados, os benefícios clínicos esperados do tratamento com hidroxiureia incluem a diminuição na frequência de episódios de dor, melhora nos valores de hemoglobina fetal e contagem de neutrófilos, redução de episódios de síndrome torácica aguda e da necessidade de transfusões sanguínea. ESTRATÉGIA DE BUSCA: Uma busca foi realizada no banco de dados eletrônico ClinicalTrials.gov, no dia 28 de março de 2022. O termo empregado foi "Sickle cell" visando a maior sensibilidade da busca. Foram considerados os ensaios clínicos de fase 2/3 e 3 de avaliação de medicamento para o tratamento da doença falciforme. Dos 893 cadastros de estudos, 92 atendiam ao critério de elegibilidade referente à fase da pesquisa. Destes, os estudos em andamento ou concluídos nos últimos cinco anos foram selecionados, restando 35 registros. Desses registros, foram excluídos aqueles que não envolviam medicamentos ou não estavam relacionados à causa-base da doença. Também foram excluídos registros envolvendo o medicamento hidroxiureia, uma vez que esse tratamento já está disponível no SUS. Deste modo, sete substâncias foram selecionadas. Uma oitava tecnologia, a L-glutamina, foi incluída por ter recebido autorização recente da FDA para a indicação abordada neste documento, embora seus registros de estudos não se enquadrem nos critérios de fase ou tempo de início e conclusão em cinco anos. MEDICAMENTOS: TECNOLOGIAS NOVAS: L-glutamina: A L-glutamina é um precursor para a síntese de glutationa, nicotinamida adenina dinucleotídeo (NAD) e arginina, substâncias que protegem os eritrócitos do dano oxidativo. No organismo, o NAD está presente em uma forma oxidada (NAD+) e reduzida (NADH). Ele é um cofator de oxidorredução (redox) onipresente nas hemácias e desempenha um papel central na manutenção do equilíbrio redox. É sabido que o estresse oxidativo contribui na fisiopatologia da doença falciforme. Crizanlizumabe: O crizanlizumabe é um anticorpo monoclonal humanizado seletivo de IgG2 kappa que promove a redução da frequência de CVOs e a inibição das interações multicelulares adesivas mediadas pela P-selectina. Esse medicamento está registrado nas agências pesquisadas, com a indicação para pacientes adultos e pediátricos a partir de 16 anos de idade com doença falciforme. Sua eficácia e segurança foram avaliadas no estudo pivotal SUSTAIN (NCT01895361), um estudo clínico de fase 2, multicêntrico, duplo-cego, randomizado e controlado por placebo, que avaliou 198 pacientes com doença falciforme e com histórico de CVOs, por um período de 52 semanas. Voxelotor: O voxelotor é um inibidor da polimerização da HbS, desenvolvido para o tratamento da doença falciforme em adultos e crianças. No FDA está indicado para crianças de 4 a 11 anos. Na EMA está indicado como opção terapêutica em pacientes a partir de 12 anos de idade. O HOPE (NCT03036813), estudo que embasou os registros nas agências EMA e FDA é um estudo de fase 3, multicêntrico, duplo-cego, randomizado e controlado por placebo, projetado para avaliar a eficácia e segurança do voxelotor. TECNOLOGIAS EMERGENTES: LentiGlobin BB305 (bb1111): A terapia gênica lovotibeglogene autotemcel (LentiGlobin; bb1111), desenvolvida pela bluebird bio®, é um tratamento experimental para a doença falciforme. Até a última atualização deste informe, não havia registro deste medicamento nas agências regulatórias pesquisadas. O FDA, no entanto, concedeu designação de medicamento órfão, fast track, terapia avançada de medicina regenerativa e de doença pediátrica rara para o lovotibeglogene autotemcel para doença falciforme. CTX-001: A CTX-001 é uma terapia celular geneticamente modificada investigada como tratamento potencial voltado para doenças de causas genéticas. O medicamento recebeu designação de droga órfã pela EMA53 e FDA, que também concedeu designação de terapia avançada de medicina regenerativa e fast track54,55. Até a última atualização desse informe, o medicamento não havia recebido registro nas agências pesquisadas. Etavopivat: O etavopivat é um ativador da piruvato quinase eritrocitária (PKR), que está sendo investigado pela Forma Therapeutics® como terapia modificadora de doença para o tratamento da doença falciforme e de outras hemoglobinopatias. Que recebeu designação de droga órfã pelas agências EMA e FDA e fast track pelo FDA60,61. Até a última atualização deste informe, a terapia não possuía registro nas agências pesquisadas. Inclacumabe: O inclacumabe é um anticorpo monoclonal IgG4 totalmente humanizado investigado para a potencial redução da frequência de CVOs causadas pela doença falciforme e internações hospitalares associadas. O medicamento promove a diminuição da formação de agregados plaquetas-leucócitos mediada pela p-selectina, reduzindo a frequência e gravidade das CVOs63,64 . Até a última atualização deste informe, o inclacumabe ainda não possuía registro nas agências FDA e EMA. Mitapivat: O mitapivat é um ativador de piruvato quinase, que recebeu designação de droga órfã pelo FDA para o tratamento da doença falciforme. A ação do mitapivat para o tratamento da doença falciforme baseia-se na ativação alostérica da piruvato quinase de eritrócitos, promovendo a redução dos níveis de 2,3-DPG e da falcização das hemácias. Os resultados sobre o mitapivat publicados até o momento são referentes segurança e tolerabilidade em diferentes doses, obtidos através de um estudo de fase 1, intervencional e não randomizado (NCT04000165), realizado em 17 indivíduos entre 18 e 70 anos de idade, com diagnóstico confirmado de anemia falciforme. Estudos em andamento Os dados dos estudos em andamento e sem resultados publicados sobre o LentiGlobin BB305, crizanlizumabe, voxelotor, etavopivat, inclacumabe e mitapivat. CONSIDERAÇÕES FINAIS: Crizanlizumabe (SEG101), L-glutamina e voxelotor são as três tecnologias recentemente aprovadas pelas agências FDA e/ou EMA para o tratamento da doença falciforme. Esses medicamentos estão associados à boa tolerabilidade apesar da alta incidência de EAs. Seus benefícios clínicos relacionam-se à diminuição de crises de dor, CVOs e aumento dos níveis de hemoglobina. As evidências acerca das demais tecnologias discutidas neste informe devem ser analisadas com cautela uma vez que, mesmo aparentemente bem toleradas e com resultados clínicos que demonstram melhora no quadro da doença, precisam de resultados mais robustos para que seja possível estabelecer um perfil concreto a respeito da segurança e benefícios esperados. Para que ocorra a oferta desses medicamentos no SUS, é necessária a análise pela Conitec, conforme disposto na Lei nº 12.401/2011, que alterou a Lei nº 8.080/1990. Os relatórios de recomendação da Conitec levam em consideração as evidências científicas sobre eficácia, a acurácia, a efetividade e a segurança do medicamento, e, também, a avaliação econômica comparativa dos benefícios e dos custos em relação às tecnologias já incorporadas e o impacto da incorporação da tecnologia no SUS.

Onasemnogene Abeparvovec (Zolgensma®) en Atrofia Muscular Espinal: (marzo 2022) / Onasemnogene Abeparvovec (Zolgensma®) in Spinal Muscular Atrophy: (March 2022)

CONTEXTO: La Atrofia Muscular Espinal (AME) engloba un grupo heterogéneo de enfermedades genéticas hereditarias caracterizadas por la degeneración y pérdida de las neuronas motoras de la medula espinal. Con una incidencia de 1 cada 25.000 nacidos vivos constituye la principal causa de mortalidade infantil por una enfermedad genética.1,2 En Argentina se estimaban 260 casos de AME para el 2019, con 31 casos nuevos por año, siendo considerada una enfermedad poco frecuente. Es una enfermedad autosómica recesiva provocada por la deleción/mutación homocigota del gen de sobrevida de la motoneurona 1 (SMN, del inglés Survival of Motor Neuron) localizado en el cromosoma 5q.2 Siendo el 94% de las veces una deleción homocigóticas del exón siete. Si bien se desconoce la función de la proteína transcripta por el gen, parecería estar involucrada en múltiples funciones esenciales de las neuronas motoras (metabolismo del ARN, su procesamiento y empalme, apoptosis y transporte axonal). TECNOLOGÍA: Onasemnogene abeparvovec (Zolgensma®) es una terapia génica recombinante que utiliza un vírus adenoasociado (VAA9) no replicativo para entregar una copia del gen SMN1 que codifica la proteína SMN humana. OBJETIVO: El objetivo del presente informe es evaluar la evidencia disponible acerca de la eficacia, seguridad y aspectos relacionados a las políticas de cobertura del uso de onasemnogene abeparvovec para AME. MÉTODOS: Se realizó una búsqueda en las principales bases de datos bibliográficas, en buscadores genéricos de internet, y financiadores de salud. Se priorizó la inclusión de revisiones sistemáticas (RS), ensayos clínicos controlados aleatorizados (ECAs), evaluaciones de tecnologías sanitarias (ETS), evaluaciones económicas, guías de práctica clínica (GPC) y políticas de cobertura de diferentes sistemas de salud. RESULTADOS: Se incluyeron dos comparaciones indirectas, ocho estudios observacionales, un reporte de congreso, tres evaluaciones de tecnología, cinco GPC, una RS de EE, una EE, y 12 informes de políticas de cobertura de tecnología para indicación. CONCLUSIONES: Evidencia de baja calidad sugiere que onasemnogene abeparvovec (Zolgensma®) puede producir un beneficio neto mayor al generar una sobrevida libre de asistencia ventilatoria permanente y mejoras en los hitos motores en niños con Atrofia Muscular Espinal (AME) tipo I con una mutación bialélica de seis meses o menos poco severos (que no requiera asistencia para la alimentación o assistência ventilatoria permanente) en comparación al tratamiento de sostén. Evidencia de baja calidad, proveniente de evidencia indirecta, sugiere que onasemnogene abeparvovec puede producir un beneficio neto considerable, al mejorar la sobrevida libre de asistencia ventilatoria permanente y los hitos motores en comparación a nursinersen en esta población aunque no genere un aumento en la sobrevida. Evidencia de muy baja calidad no permite concluir la efectividad del tratamiento en niños mayores de seis meses. Guías de practica clínica de España, Europa y Alemania no son concluyentes para dar uma recomendación a favor o en contra de la implementación de onasemnogene abeparvovec, sin embargo, mencionan recomendaciones para la administración y seguimiento. Una guía de práctica clínica del Hospital "Prof. Dr. Juan P. Garrahan" en Argentina, menciona la tecnología como emergente en el tratamiento de AME, pero no realiza recomendaciones. Cabe aclarar que su publicación fue muy próxima a la aprobación de onasemnogene abeparvovec por la Administración de Alimentos y Medicamentos de los Estados Unidos. Las políticas de cobertura de Latinoamérica, Australia y Estados Unidos (del sistema público) no la mencionan. Canadá y el Reino Unido establecen cobertura en base a un acuerdo de precios. Canadá solo en menores de seis meses y el Reino Unido en menores de siete meses, estableciendo la necesidad de la evaluación de un equipo multidisciplinario nacional en niños entre siete y 12 meses. Políticas de cobertura de financiadores privados de Estados Unidos cubren de onasemnogene abeparvovec en AME tipo I en niños menores de dos años que cumplan ciertos requisitos. No se encontraron estudios de costo­efectividad en Argentina, de acuerdo al impacto presupuestario estimado en el país se establece que es un tratamiento de alto costo. Datos de evaluaciones económicas en otras regiones del mundo sugieren que no seria costo­efectivo en comparación al tratamiento de sostén para Argentina. Debido a la falta de evidencia de buena calidad y análisis de costo­efectividad en la comparación entre onasemnogene abeparvovec y nursinersen no se puede recomendar un tratamiento sobre otro, por lo cual se deben contemplar otros factores, como costos y acceso, para llegado el caso, determinar la elección entre distintas alternativas terapéuticas.

In Vitro and In Vivo Evaluation of Human Adenovirus Type 49 as a Vector for Therapeutic Applications.

The human adenovirus phylogenetic tree is split across seven species (A-G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of pre-existing immunity detected across screened populations. However, many aspects of the basic virology of species D-such as their cellular tropism, receptor usage, and in vivo biodistribution profile-remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49)-a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry, but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting, whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells, and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen, whilst avoiding liver interactions, such as intravascular vaccine applications.

Limitations and advances in new treatments and future perspectives of corneal blindness / Limitações e avanços em novos tratamentos e perspectivas futuras na cegueira corneal

ABSTRACT This review is intended to describe the therapeutic approaches for corneal blindness, detailing the steps and elements involved in corneal wound healing. It also presents the limitations of the actual surgical and pharmacological strategies used to restore and maintain corneal transparency in terms of long-term survival and geographic coverage. In addition, we critically review the perspectives of anabolic agents, including vitamin A, hormones, growth factors, and novel promitotic and anti-inflammatory modulators, to assist corneal wound healing. We discuss the studies involving nanotechnology, gene therapy, and tissue reengineering as potential future strategies to work solely or in combination with corneal surgery to prevent or revert corneal blindness.(AU)

RESUMO O presente trabalho traz uma revisão das abordagens terapêuticas para a cegueira da córnea. O estudo detalha as etapas e os elementos envolvidos na cicatrização da córnea. Ele mostra as limitações das estratégias cirúrgicas e farmacológicas usadas para restaurar e manter a transparência da córnea em termos de sobrevida a longo prazo e alcance geográfico. As perspectivas dos agentes anabólicos, incluindo vitamina A, hormônios, fatores de crescimento e novos moduladores pró-mitóticos e anti-inflamatórios para auxiliar a cicatrização da ferida na córnea, são revisadas criticamente. Aqui, apresentamos estudos envolvendo nanotecnologia, terapia gênica e reengenharia de tecidos como possíveis estratégias futuras para atuar de maneira isolada ou combinada com a cirurgia da córnea para prevenir ou reverter a cegueira corneana.(AU)

Title: Gene transfer by pyro-drive jet injector is a novel therapeutic approach for muscle diseases.

The angiogenic gene therapy is an attractive approach for the treatment of ischemic muscle diseases, including peripheral arterial disease and ischemic heart diseases. Although a variety of gene transfer methods have been developed, the efficiency of gene transfer is still limited. We have been developing the needleless high-energy bioinjector device, Pyro-drive Jet Injector (PJI), based on pyrotechnics using a combination of ignition powder and gunpowder, however, the utility of PJI in gene transfer into muscle tissues remains unclear. pcDNA3.1 plasmid containing Flag was injected to the thigh muscles of C57BL/6J mice using PJI or needle, as a control. Histological analysis demonstrated that the protein expression of Flag was observed in a wider range in PJI group than in needle group. To assess the validity of PJI for gene therapy, pcDNA3.1-human fibroblast growth factor 2 (FGF2), which has angiogenic activity and tissue protective properties, was injected into the ischemic thigh muscles with PJI or needle. ELISA assay revealed that the protein expression of FGF2 was increased in the thigh muscle tissues by PJI-mediated gene delivery. Significantly, histological analyses revealed that muscle fiber cross-sectional area and the number of endothelial marker CD31 (+) cells was increased in ischemic hind-limb tissues of the PJI-FGF2 group but not in those of needle-FGF2 group. To expand the applicability of the PJI-mediated gene transfer, pcDNA3.1-venus plasmid was injected into murine hearts with PJI or needle. PJI method was successful in gene transfer into murine hearts, especially into cardiomyocytes, with high efficiency when compared to needle method. Collectively, the non-needle, non-liposomal and non-viral gene transfer by PJI could be a novel therapeutic approach for muscle diseases.

Onasemnogene abeparvovec (Zolgensma®) en atrofia muscular espinal / Onasemnogene abeparvovec (Zolgensma®) in spinal muscular atrophy

INTRODUCCIÓN: La atrofia muscular espinal (AME) es una afección neuromuscular hereditaria que afecta las células nerviosas (neuronas motoras) en un área de la médula espinal llamada cuerno anterior. Las personas con AME tienen un gen faltante o mutado de la neurona motora de supervivencia 1 (SMN1), localizado en la región cromosómica 5q1 y que produce una proteína llamada proteína de la neurona motora de supervivencia (SMN). El locus AME está duplicado y en la parte más central de este locus existe un gen homólogo conocido como SMN2, a partir de cuya transcripción también puede ser sintetizada la proteína SMN. Todos los pacientes con AME presentan como signos clínicos debilidad generalizada a predominio proximal y de miembros inferiores, reflejos osteotendinosos disminuidos o ausentes, y compromiso de músculos intercostales con relativa preservación del diafragma. Esto deriva en problemas con la respiración, así como con las actividades de movimiento (motoras) como gatear, sentarse, caminar, alimentarse y controlar la cabeza; sin embargo, la capacidad intelectual no se ve afectada. La incidencia mundial de AME al nacer es de alrededor de 1 en 10.000, con una tasa de portadores entre 1/35 y 1/50, por lo tanto, se clasifica como una enfermedad poco frecuente, y se encuentra subdividida en los distintos tipos clínicos (I, II, III, IV) asociados a la presencia de la proteína SMN o su número de copias. La sobrevida de la forma con mayor incidencia (AME tipo I) varía entre el 30 a 70% al año de vida. Actualmente, no existe un tratamiento curativo para AME y sólo se dispone de tratamiento sintomático para retrasar la progresión de la enfermedad y sus efectos incapacitantes, así como el tratamiento de sostén nutricional, ventilatorio y neuromuscular para mitigar sus complicaciones. La única terapia farmacológica extendida a nivel mundial a la fecha para el tratamiento de AME es nusinersen (Spinraza®), siendo onasemnogene abeparvovec (Zolgensma®) una nueva terapia génica recombinante recientemente aprobada en Estados Unidos y Europa. OBJETIVO: El objetivo del presente informe es evaluar la eficacia, seguridad, políticas de cobertura y aspectos económicos de Zolgensma® en AME. MÉTODOS: BÚSQUEDA BIBLIOGRÁFICA Se buscó en los sitios públicos de Pubmed, LILACS, BRISA/REDETSA-, CRD (del inglés Centre for Reviews and Dissemination- University of York), Cochrane; en "buscadores genéricos de internet" y sociedades científicas. En lo que respecta a agencias de ETS, se buscó en: NICE (del inglés, National Institute for Health and Clinical Excellence) del Reino Unido; PBAC (del inglés, The Pharmaceutical Benefits Advisory Committee) de Australia; CADTH (del inglés, Canadian Agency for Drugs and Technologies in Health) de Canadá y CONITEC (Comissão Nacional de Incorporação de Tecnologías no SUS) de Brasil. RESULTADOS: Nuestra revisión arrojó que existe evidencia de baja calidad (fundamentalmente debida a estudios abiertos, con bajo número de pacientes y seguimientos abreviados) que sugiere que en bebés sintomáticos con AME tipo I, con deleciones homocigotas de SMN1, dos copias de SMN2 y ninguna mutación modificadora c.859G>C de SMN2, el tratamiento con Zolgensma® frente al tratamiento de sostén mejoraría de forma importante la supervivencia sin necesidad de asistencia ventilatoria a los 14 meses de vida (seguimiento = 10 meses), con desarrollo de habilidad motora a 23 meses de seguimiento (aunque evidencia de muy baja calidad no permite concluir si el tratamiento con Zolgensma® frente al tratamiento de sostén mejoraría de forma importante la puntuación de CHOP-INTEND como escala validada). Por otro lado, evidencia de muy baja calidad no permite concluir sobre la eficacia y seguridad de la comparación de Zolgensma® frente a Spinraza® (no existen estudios cabeza a cabeza), ni sobre Zolgensma® a un seguimiento más prolongado en bebés sintomáticos con AME tipo I, como tampoco en bebés asintomáticos con AME tipo I. No se hallaron evaluaciones económicas publicadas para Argentina: sin embargo, evaluaciones provenientes de Estados Unidos sugieren que el tratamiento con Zolgensma® sería costo-efectivo frente a Spinraza® o al tratamiento de sostén para un umbral especial (aunque considerando esquemas de costos prestacionales sensiblemente diferentes a nuestro país). Un análisis de impacto presupuestario de elaboración propia estimó que el costo incremental neto de incluir Zolgensma® a nuestro sistema de salud tendría un elevado impacto sobre el financiamiento, sin generar ahorros prestacionales ni beneficios relevantes. CONCLUSIÓN: Según el presente informe, el análisis de Zolgensma® requiere la consideración de precios ajustados a las características y posibilidades del sistema de salud de nuestro país, con una alta selección de la población objetivo y mecanismos de generación de mayor evidencia prospectiva a cargo de los productores de la tecnología. Finalmente, no se hallaron guías de práctica clínica que recomienden específicamente el tratamiento con Zolgensma®, aunque sí se hallaron recomendaciones generales para la implementación del mismo. La totalidad de los países de Latinoamérica relevados no dan cobertura al tratamiento con Zolgensma®, teniendo sólo cobertura en algunos países de altos ingresos (Estados Unidos y Francia).

Polyprenol-Based Lipofecting Agents for In Vivo Delivery of Therapeutic DNA to Treat Hypertensive Rats.

Development of efficient vectors for transfection is one of the major challenges in genetic engineering. Previous research demonstrated that cationic derivatives of polyisoprenoids (PTAI) may serve as carriers of nucleic acids. In the present study, the effectiveness of two PTAI-based formulations (PTAI-6-8 and 10-14) was investigated and compared to the commercial reagents. The purpose of applied gene therapy was to enhance the expression of vascular endothelial growth factor (VEGF-A) in the renal medulla of spontaneously hypertensive rats (SHR) and to test its potential as a novel antihypertensive intervention. In the first part of the study (in vitro), we confirmed that PTAI-based lipoplexes efficiently transfect XC rat sarcoma cells and are stable in 37 °C for 7 days. In the in vivo experiments, we administered selected lipoplexes directly to the kidneys of conscious SHR (via osmotic pumps). There were no blood pressure changes and VEGF-A level in renal medulla was significantly higher only for PTAI-10-14-based formulation. In conclusion, despite the promising results, we were not able to achieve VEGF-A expression level high enough to verify VEGF-A gene therapy usefulness in SHR. However, results of our study give important indications for the future development of PTAI-based DNA carriers and kidney-targeted gene delivery.

Advances in intelligent DNA nanomachines for targeted cancer therapy.

As an emerging field, DNA nanotechnology has been applied to the fabrication of drug delivery systems. Unprecedented spatial addressability and intrinsic sequence encoding enable DNA strands to self-assemble into well-defined 2D and 3D DNA nanostructures with specifically controlled sizes, shapes and surface charges. Multifunctional DNA nanostructures have been created and applied as promising platforms for drug delivery, imaging, and theranostics. Advantages of chemotherapy, gene therapy, and immunotherapy, among others, have been integrated into such functional nanodevices, showing potential in tumor-targeted therapy and diagnosis. In this review, we summarize general methods for the construction of DNA nanodevices and focus on targeting strategies favored by the compatibility of DNA nanotechnology. Additionally, we highlight the outlook and challenges facing the use of DNA nanotechnology in cancer therapy.

Historic Overview of Genetic Engineering Technologies for Human Gene Therapy.

The concepts of gene therapy were initially introduced during the 1960s. Since the early 1990s, more than 1900 clinical trials have been conducted for the treatment of genetic diseases and cancers mainly using viral vectors. Although a variety of methods have also been performed for the treatment of malignant gliomas, it has been difficult to target invasive glioma cells. To overcome this problem, immortalized neural stem cell (NSC) and a nonlytic, amphotropic retroviral replicating vector (RRV) have attracted attention for gene delivery to invasive glioma. Recently, genome editing technology targeting insertions at site-specific locations has advanced; in particular, the clustered regularly interspaced palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) has been developed. Since 2015, more than 30 clinical trials have been conducted using genome editing technologies, and the results have shown the potential to achieve positive patient outcomes. Gene therapy using CRISPR technologies for the treatment of a wide range of diseases is expected to continuously advance well into the future.

Acoustofluidic sonoporation for gene delivery to human hematopoietic stem and progenitor cells.

Advances in gene editing are leading to new medical interventions where patients' own cells are used for stem cell therapies and immunotherapies. One of the key limitations to translating these treatments to the clinic is the need for scalable technologies for engineering cells efficiently and safely. Toward this goal, microfluidic strategies to induce membrane pores and permeability have emerged as promising techniques to deliver biomolecular cargo into cells. As these technologies continue to mature, there is a need to achieve efficient, safe, nontoxic, fast, and economical processing of clinically relevant cell types. We demonstrate an acoustofluidic sonoporation method to deliver plasmids to immortalized and primary human cell types, based on pore formation and permeabilization of cell membranes with acoustic waves. This acoustofluidic-mediated approach achieves fast and efficient intracellular delivery of an enhanced green fluorescent protein-expressing plasmid to cells at a scalable throughput of 200,000 cells/min in a single channel. Analyses of intracellular delivery and nuclear membrane rupture revealed mechanisms underlying acoustofluidic delivery and successful gene expression. Our studies show that acoustofluidic technologies are promising platforms for gene delivery and a useful tool for investigating membrane repair.

Targeted delivery of engineered auditory sensing protein for ultrasound neuromodulation in the brain.

Sonogenetics is a promising approach for in vivo neuromodulation using ultrasound (US) to non-invasively stimulate cells in deep tissue. However, sonogenetics requires accurate transduction of US-responsive proteins into target cells. Here, we introduce a non-invasive and non-viral approach for intracerebral gene delivery. This approach utilizes temporary ultrasonic disruption of the blood-brain barrier (BBB) to transfect neurons at specific sites in the brain via DNA that encodes engineered US-responsive protein (murine Prestin (N7T, N308S))-loaded microbubbles (pPrestin-MBs). Prestin is a transmembrane protein that exists in the mammalian auditory system and functions as an electromechanical transducer. We further improved the US sensitivity of Prestin by introducing specific amino acid substitutions that frequently occur in sonar species into the mouse Prestin protein. We demonstrated this concept in mice using US with pPrestin-MBs to non-invasively modify and activate neurons within the brain for spatiotemporal neuromodulation. Method: MBs composed of cationic phospholipid and C3F8 loaded with mouse Prestin plasmid (pPrestin) via electrostatic interactions. The mean concentration and size of the pPrestin-MBs were (16.0 ± 0.2) × 109 MBs/mL and 1.1 ± 0.2 µm, respectively. SH-SY5Y neuron-like cells and C57BL mice were used in this study. We evaluated the gene transfection efficiency and BBB-opening region resulting from pPrestin-MBs with 1-MHz US (pressure = 0.1-0.5 MPa, cycle = 50-10000, pulse repetition frequency (PRF): 0.5-5 Hz, sonication time = 60 s) using green fluorescence protein (Venus) and Evans blue staining. Results: The maximum pPrestin expression with the highest cell viability occurred at a pressure of 0.5 MPa, cycle number of 5000, and PRF of 1 Hz. The cellular transfection rate with pPrestin-MBs and US was 20.2 ± 2.5%, which was 1.5-fold higher than that of commercial transfection agents (LT-1). In vivo data suggested that the most profound expression of pPrestin occurred at 2 days after performing pPrestin-MBs with US (0.5 MPa, 240 s sonication time). In addition, no server erythrocyte extravasations and apoptosis cells were observed at US-sonicated region. We further found that with 0.5-MHz US stimulation, cells with Prestin expression were 6-fold more likely to exhibit c-Fos staining than cells without Prestin expression. Conclusion: Successful activation of Prestin-expressing neurons suggests that this technology provides non-invasive and spatially precise selective modulation of one or multiple specific brain regions.

Fe3+-Coordinated Multifunctional Elastic Nanoplatform for Effective in Vivo Gene Transfection.

The common phenomenon that the nonviral vectors have much lower transfection efficiency in vivo than in vitro greatly restricts their further developments and applications. Possible reasons are lacking targeting ability, elimination by the reticuloendothelial system (RES), and insufficient nuclear transport. Here, a novel, flexible, and deformable polymer Fe@PEI-R12 (tLyp-1-NLS) is reported for shortening the gap between in vitro and in vivo gene transfection efficiency. The amorphous network structure Fe@PEI with deformation ability acquired by coordination cross-linking of Fe3+ and low-molecular-weight polyethylenimine (LMW-PEI) constructs the core and serves as the gene reservoir, and it can squeeze out through RES filter holes when trapped in the spleen. The bifunctional peptide R12 provided tumor targeting and enhanced nuclear delivery ability. Additionally, the Fe3+ from Fe@PEI-R12 could trigger endogenous hydrogen peroxide (H2O2) decomposition to produce O2, thereby reducing the adverse effects of tumor hypoxia. It is demonstrated that the Fe@PEI-R12/pDNA complexes could pass through membrane filters, subsequently achieving long circulation time, and Fe@PEI-R12 had a tendency to accumulate in tumor tissue and mediate pGL3-control expression. Therefore, the multifunctional nanoplatform has the potential for effective in vivo gene delivery.

Role of Nanobiotechnology in Drug Delivery.

This chapter is a brief overview of use of nanobiotechnology in drug delivery. Several types of nanoparticles are available. Nanoparticulate formulations of normally used drugs have increased efficacy due to improved absorption and require lower dosage with less side effects than standard formulations. Nanobiotechnology also facilitates targeted drug delivery of anticancer drugs, which is important for the management of cancer. Nanoparticles also facilitate crossing of biological barriers in the human body for drug delivery to targeted organs, for example, crossing the blood-brain barrier to reach the brain. Nanobiotechnology applications in delivery of biological therapies are expanding in areas such as cell and gene therapies, siRNAs, and monoclonal antibodies. Some nanoparticles can carry more than one therapeutic molecule enabling multimodal therapy and combination with physical modalities such as radiotherapy in cancer. Nanorobotics is developing with applications in drug delivery, particularly for cancer. Other anticipated developments in this area include use of nanotechnology for creating intelligent drug release devices.

Targeted siRNA Delivery Using Lipid Nanoparticles.

Efficient intracellular delivery of small-interfering ribonucleic acid (siRNA) to the target organ or tissues in the body is assumed as the main hurdle for a widespread use of siRNAs in the clinics. Solid lipid-based nanoparticles (SLNs) and derivatives can potentially fit this purpose by enabling to overcome the extracellular and intracellular physiological barriers affecting the delivery. For that, rational formulations and rational process designs are needed. This chapter addresses a comprehensive description and critical appraisal of the main production methods of this particular type of lipid nanoparticles and the leading strategies to prompt a targeted delivery of siRNA.

Nanocarrier Mediated siRNA Delivery Targeting Stem Cell Differentiation.

Stem cell-based regenerative medicine holds exceptional therapeutic potential and hence the development of efficient techniques to enhance control over the rate of differentiation has been the focus of active research. One of the strategies to achieve this involves delivering siRNA into stem cells and exploiting the RNA interference (RNAi) mechanism. Transport of siRNA across the cell membrane is a challenge due to its anionic property, especially in primary human cells and stem cells. Moreover, naked siRNA incites immune responses, may cause off-target effects, exhibits low stability and is easily degraded by endonucleases in the bloodstream. Although siRNA delivery using viral vectors and electroporation has been used in stem cells, these methods demonstrate low transfection efficiency, cytotoxicity, immunogenicity, events of integration and may involve laborious customization. With the advent of nanotechnology, nanocarriers which act as novel gene delivery vehicles designed to overcome the problems associated with safety and practicality are being developed. The various nanomaterials that are currently being explored and discussed in this review include liposomes, carbon nanotubes, quantum dots, protein and peptide nanocarriers, magnetic nanoparticles, polymeric nanoparticles, etc. These nanodelivery agents exhibit advantages such as low immunogenic response, biocompatibility, design flexibility allowing for surface modification and functionalization, and control over the surface topography for achieving the desired rate of siRNA delivery and improved gene knockdown efficiency. This review also includes discussion on siRNA co-delivery with imaging agents, plasmid DNA, drugs etc. to achieve combined diagnostic and enhanced therapeutic functionality, both for in vitro and in vivo applications.

A nanogel based oral gene delivery system targeting SUMOylation machinery to combat gut inflammation.

Poor success rates and challenges associated with the current therapeutic strategies of inflammatory bowel disease (IBD) have accelerated the emergence of gene therapy as an alternative treatment option with great promise. However, oral delivery of nucleic acids (NAs) to an inflamed colon is challenged by multiple barriers presented by the gastrointestinal, extracellular and intracellular compartments. Therefore, we screened a series of polyaspartic acid-derived amphiphilic cationic polymers with varied hydrophobicity for their ability to deliver NAs into mammalian cells. Using the most effective TAC6 polymer, we then engineered biocompatible and stable nanogels composed of polyplexes (TAC6, NA) and an anionic polymer, sodium polyaspartate, that were able to deliver the NAs across mammalian cells using caveolae-mediated cellular uptake. We then utilized these nanogels for oral delivery of PIAS1 (protein inhibitor of activated STAT1), a SUMO 3 ligase, encoding plasmid DNA since PIAS1 is a key nodal therapeutic target for IBD due to its ability to control NF-κB-mediated inflammatory signaling. We show that plasmid delivery using TAC6-derived nanogels diminished gut inflammation in a murine colitis model. Therefore, our study presents engineering of orally deliverable nanogels that can target SUMOylation machinery to combat gut inflammation with very high efficacy.

Rapid establishment of stable retroviral packaging cells and recombinant susceptible target cell lines employing novel transposon vectors derived from Sleeping Beauty.

Viral vector particles derived from murine leukemia virus (MLV) mediate highly efficient stable gene transfer used in gene therapeutic approaches and in the generation of transgenic cell lines. However, the establishment of stable viral packaging cells (VPCs) is a time-consuming challenge. To overcome this limitation, we successfully generated novel Sleeping Beauty-derived transposon vectors entailing envelope and packaging expression cassettes as well as a transfer vector. Upon multiplexed transposition in human cells, VPC bulk populations yielding titers of over 1 × 106 transduction-competent vectors were established within three weeks. In contrast, conventional plasmid-based establishment of VPCs, conducted in parallel, took much longer and yielded significantly lower vector productivity and vector fitness. The generated MLV vectors decorated with the envelope proteins of ecotropic MLV PVC-211mc mediated efficient transduction of Chinese hamster ovary (CHO) cells. Cell susceptibility was further elevated upon recombinant expression of the murine ecotropic receptor mCAT employing a transposon vector.

Tetramer-Based Enrichment of Preexisting Anti-AAV8 CD8+ T Cells in Human Donors Allows the Detection of a TEMRA Subpopulation.

Pre-existing immunity to AAV capsid may compromise the safety and efficiency of rAAV-mediated gene transfer in patients. Anti-capsid cytotoxic immune responses have proven to be a challenge to characterize because of the scarcity of circulating AAV-specific CD8+ T lymphocytes which can seldom be detected with conventional flow cytometry or ELISpot assays. Here, we used fluorescent MHC class I tetramers combined with magnetic enrichment to detect and phenotype AAV8-specific CD8+ T cells in human PBMCs without prior amplification. We showed that all healthy individuals tested carried a pool of AAV8-specific CD8+ T cells with a CD45RA+ CCR7- terminally-differentiated effector memory cell (TEMRA) fraction. Ex vivo frequencies of total AAV-specific CD8+ T cells were not predictive of IFNγ ELISpot responses but interestingly we evidenced a correlation between the proportion of TEMRA cells and IFNγ ELISpot positive responses. TEMRA cells may then play a role in recombinant AAV-mediated cytotoxicity in patients with preexisting immunity. Overall, our results encourage the development of new methods combining increased detection sensitivity of AAV-specific T cells and their poly-functional assessment to better characterize and monitor AAV capsid-specific cellular immune responses in the perspective of rAAV-mediated clinical trials.

Infectivity of adeno-associated virus serotypes in mouse testis.

BACKGROUND: Recombinant adeno-associated viruses (AAVs) are emerging as favoured transgene delivery vectors for both research applications and gene therapy. In this context, a thorough investigation of the potential of various AAV serotypes to transduce specific cell types is valuable. Here, we rigorously tested the infectivity of a number of AAV serotypes in murine testis by direct testicular injection. RESULTS: We report the tropism of serotypes AAV2, 5, 8, 9 and AAVrh10 in mouse testis. We reveal unique infectivity of AAV2 and AAV9, which preferentially target intertubular testosterone-producing Leydig cells. Remarkably, AAV2 TM, a mutant for capsid designed to increase transduction, displayed a dramatic alteration in tropism; it infiltrated seminiferous tubules unlike wildtype AAV2 and transduced Sertoli cells. However, none of the AAVs tested infected spermatogonial cells. CONCLUSIONS: In spite of direct testicular injection, none of the tested AAVs appeared to infect sperm progenitors as assayed by reporter expression. This lends support to the current view that AAVs are safe gene-therapy vehicles. However, testing the presence of rAAV genomic DNA in germ cells is necessary to assess the risk of individual serotypes.

TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells.

CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as ß-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex, consisting of purified Cas9 protein and guide RNA, into HSPCs. Because ß-hemoglobinopathies are most prevalent in developing countries, it is desirable to have a reliable, efficient, easy-to-use and cost effective delivery method. With this goal in mind, we developed TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF), a new method to quickly and effectively deliver RNPs into HSPCs by passing a RNP and cell mixture through a filter membrane. We achieved robust gene editing in HSPCs using TRIAMF and demonstrated that the multilineage colony forming capacities and the competence for engraftment in immunocompromised mice of HSPCs were preserved post TRIAMF treatment. TRIAMF is a custom designed system using inexpensive components and has the capacity to process HSPCs at clinical scale.