Objectives
Exploiting the forces of human T cells for treatment has led to the current paradigm of emerging immunotherapy strategies. Genetic engineering of the T‐cell receptor (TCR) redirects ...specificity, ablates alloreactivity and brings significant progress and off‐the‐shelf options to emerging adoptive T‐cell transfer (ACT) approaches. Targeted CRISPR/Cas9‐mediated double‐strand breaks in the DNA enable knockout or knock‐in engineering.
Methods
Here, we perform CRISPR/Cas9‐mediated TCR knockout using a therapeutically relevant ribonucleoprotein (RNP) delivery method to assess the safety of genetically engineered T‐cell products. Whole‐genome sequencing was performed to analyse whether CRISPR/Cas9‐mediated DNA double‐strand break at the TCR locus is associated with off‐target events in human primary T cells.
Results
TCRα chain and TCRβ chain knockout leads to high on‐target InDel frequency and functional knockout. None of the predicted off‐target sites could be confirmed experimentally, whereas whole‐genome sequencing and manual Integrative Genomics Viewer (IGV) review revealed 9 potential low‐frequency off‐target events genome‐wide. Subsequent amplification and targeted deep sequencing in 7 of 7 evaluable loci did not confirm these low‐frequency InDels. Therefore, off‐target events are unlikely to be caused by the CRISPR/Cas9 engineering.
Conclusion
The combinatorial approach of whole‐genome sequencing and targeted deep sequencing confirmed highly specific genetic engineering using CRISPR/Cas9‐mediated TCR knockout without potentially harmful exonic off‐target effects.
Exploiting forces of T cells for treatment through genetic engineering of the T‐cell receptor (TCR) redirects specificity, ablates alloreactivity and brings significant progress and off‐the‐shelf options. CRISPR/Cas9‐mediated double‐strand breaks in the TCRα and TCRβ chain are supposed to be a site‐specific TCR knockout. Whole‐genome sequencing and targeted deep sequencing confirmed highly specific DNA engineering without potentially harmful exonic off‐target effects in primary human T cells.
Evolution of Epiphysiodesis Surgery Sepúlveda O, Matías; Ferrada A, Paulina; Miranda-Chacón, Zaray ...
Andes pediatrica : revista Chilena de pediatría
92, Številka:
5
Journal Article
Recenzirano
Surgical modulation of physeal growth, currently better known as "Guided Growth", is a procedure used as a treatment for length differences or axis alterations of the extremities. Although its use is ...increasing due to the development of new less invasive techniques, there are no statistical analyzes in the literature that support this.
To evaluate the incidence of the surgery codified as "Epi- physiodesis (Femur and/or Tibia)" in the Chilean population, from January 1st, 2010 to December 31, 2019, the days of hospital stay associated with that code and the demographic data of the analyzed po pulation.
We obtained from the Chilean Department of Statistics and Health Information (DEIS) database, all hospital discharges from January 1st, 2010 to December 31, 2019, from which we extracted the results with the code "Epiphysiodesis (Femur and/or Tibia)", in children under 15 years. The number of procedures per year, days of hospital stay, age, and sex were analyzed.
In Chile, the number of procedures coded as "Epiphysiodesis (Femur and/or Tibia)" increa sed during the period analyzed. The days of hospital stay decreased progressively between 2012 and 2017. The procedure was performed mostly in women, and in the 5-9 years age group, showing an upward trend in children of the group of 10-14 years between 2015 and 2019.
Further analysis must be carried out to determine the factors that produce these results, which are probably related to access to more efficient and simple techniques, with less morbidity.
Resumen: El crecimiento guiado es una opción quirúrgica de uso creciente para la corrección de las deformidades angulares de extremidades inferiores en pacientes esqueléticamente inmaduros. Es ...posible de realizar en múltiples planos o segmentos, con excelentes resultados. Su uso más frecuente es en deformidades del plano coronal alrededor de la rodilla. La epifisiodesis permanente se puede realizar en pacientes dentro de los dos años previos al término del crecimiento longitudinal del segmento a tratar, considerando la epifisiodesis temporal para los pacientes con más de 2 años de crecimiento restante.En casos leves a moderados las tasas de éxito llegan incluso al 100% en algunas series, en tanto, pacientes con enfermedad de Blount, obesidad, edad esquelética avanzada o deformidades severas, tienen menos posibilidades de lograr una corrección completa.Independientemente de la técnica quirúrgica, es necesario una adecuada planificación preoperatoria, educación familiar y un seguimiento estricto para así minimizar las complicaciones y permitir una excelente corrección de la deformidad con una morbilidad mínima. Summary: Guided Growth is a surgical option of increasing use for the correction of angular deformities of the lower extremities in skeletally immature patients. It is possible to perform in multiple planes or segments, with excellent results. Its most frequent use is in deformities of the coronal plane around the knee. Permanent epiphysiodesis can be performed in patients within 2 years before the end of longitudinal growth of the segment to be treated, considering temporary epiphysiodesis for patients with more than 2 years of remaining growth.In mild to moderate cases, the success rates reach even 100% in some series, while patients with Blount's disease, obesity, advanced skeletal age or severe deformities are less likely to achieve a complete correction.Regardless of the surgical technique, adequate preoperative planning, family education and strict follow-up are necessary to minimize complications and allow excellent correction of the deformity with minimal morbidity.
CRECIMIENTO GUIADO EN EXTREMIDADES INFERIORES Sepúlveda, Matías Felipe; Ferrada, Paulina
Revista Médica Clínica Las Condes,
May-June 2021, 2021-05-00, Letnik:
32, Številka:
3
Journal Article
Recenzirano
Odprti dostop
El crecimiento guiado es una opción quirúrgica de uso creciente para la corrección de las deformidades angulares de extremidades inferiores en pacientes esqueléticamente inmaduros. Es posible de ...realizar en múltiples planos o segmentos, con excelentes resultados. Su uso más frecuente es en deformidades del plano coronal alrededor de la rodilla. La epifisiodesis permanente se puede realizar en pacientes dentro de los dos años previos al término del crecimiento longitudinal del segmento a tratar, considerando la epifisiodesis temporal para los pacientes con más de 2 años de crecimiento restante.
En casos leves a moderados las tasas de éxito llegan incluso al 100% en algunas series, en tanto, pacientes con enfermedad de Blount, obesidad, edad esquelética avanzada o deformidades severas, tienen menos posibilidades de lograr una corrección completa.
Independientemente de la técnica quirúrgica, es necesario una adecuada planificación preoperatoria, educación familiar y un seguimiento estricto para así minimizar las complicaciones y permitir una excelente corrección de la deformidad con una morbilidad mínima.
Guided Growth is a surgical option of increasing use for the correction of angular deformities of the lower extremities in skeletally immature patients. It is possible to perform in multiple planes or segments, with excellent results. Its most frequent use is in deformities of the coronal plane around the knee. Permanent epiphysiodesis can be performed in patients within 2 years before the end of longitudinal growth of the segment to be treated, considering temporary epiphysiodesis for patients with more than 2 years of remaining growth.
In mild to moderate cases, the success rates reach even 100% in some series, while patients with Blount's disease, obesity, advanced skeletal age or severe deformities are less likely to achieve a complete correction.
Regardless of the surgical technique, adequate preoperative planning, family education and strict follow-up are necessary to minimize complications and allow excellent correction of the deformity with minimal morbidity.
CONCLUSION While synthetic immunotherapy has been introduced into the standard of care treatment of patients with B-lineage malignancies, patients with T-lineage malignancies have not gained benefit ...from this novel approach, yet. The main reason is the challenging choice of target antigen in T-lineage malignancies such as pediatric T cell acute lymphoblastic leukemia (T-ALL). As activating mutations of CD28 have already been described in T cell malignancies, we set out to quantify surface expression of CD28 and other costimulatory molecules on primary pediatric T-ALL samples and explore the utility of this functionally relevant molecule as a novel target antigen. Bone marrow (BM) samples of pediatric T-ALL patients (n=54) at time of diagnosis and healthy control individuals (n=14) were collected. We quantified surface expression of CD28 and 25 additional co-stimulatory or co-inhibitory molecules by flow cytometry after gating on T cell precursors (living singlets, CD45 dim/SSC-A low and CD7 +). Subsequently, we generated a set of 20 CD28 directed second-generation chimeric antigen receptors (CARs) based on five different monoclonal antibodies with variation in hinge domain and light chain / heavy chain chronology. We analyzed specific CAR activity against CD28 expressing T-ALL cell lines after retroviral transduction into primary human T cells. Additional CD28 knockout (KO) by CRISPR/Cas9 could prevent T cell fratricide. CAR constructs were subjected to in vivo testing in a T-ALL NSG mouse model with transplantation of 7.5e4 CCRF-CEM cells transduced with firefly luciferase on day 0 and transfer of 2.5e6 CD28 KO T cells on day 3 harboring either of both CD28 CAR molecules. We used CD7 CAR T cells as positive control, CD19 CAR T cells and CD28 KO T cells without CAR as negative controls. We observed significant upregulation of CD28 on T-ALL leukemia when compared to healthy BM donors (Figure 1A, mean 68.8% vs. 3.7%, p=0.0002). We confirmed upregulation of CD28 in published T-ALL RNA-expression data sets. Interestingly, other co-stimulatory molecules such as CD127 were upregulated, too (mean 50.0% vs. 14.6%, p=0.0001), while co-inhibitory molecules such as CD160, TIGIT and TIM-3 were strongly downregulated (>10fold and p<0.0001 each). We hypothesized that this could imply a functional relevance of CD28 in T-ALL. Therefore, we performed co-culture assays of monocyte derived dendritic cells (CDs) expressing CD28 ligands CD80 and CD86 with different T-lineage leukemia cell lines. The presence of DCs significantly increased proliferation of CD28 + T-ALL cell lines under stress conditions. In order to test the feasibility of CD28 CAR T cells, we generated CD28 KO primary T cells without CAR. When tested in co-culture assays with leukemia cells and T cell engagers, CD28 KO T cells showed unchanged cytotoxic capacity and target-dependent activation illustrating that CD28 KO T cells retain short-term effector functions. Based on cytotoxic capacity and CAR T cell expansion, we identified two lead CD28 CAR candidates out of a pool of 20 CD28 CARs. After CD28 KO, CD28 CAR-T cells showed in vitro expansion comparable to CD19 CAR T cells. Next, we asked how CD28 CAR T cells compare to CD7 CAR T cells, which are currently evaluated clinical trials for T-ALL therapy. Therefore, we generated CD7 KO T cells that were subsequently transduced with a functional CD7 CAR. In vitro, both CD28 CAR T cells and CD7 CAR T cells showed killing of CD28 +CD7 + CCRF-CEM cells of >90% at an effector:target (E:T) ratio of 0.2:1, with CD7 being outperformed by one of the CD28 CARs while outperforming the other CD28 CAR at an E:T ratio of 0.04:1 (Figure 1B). Finally, we went on to validate the functionality of CD28 CAR T cells in vivo. We observed significantly prolonged survival of mice treated with CD28 CAR T cells and CD7 CAR T cells when compared to CD19 CAR T cells or control T cells. No difference in survival between CD7 and CD28 CAR T cell treated mice could be observed in two independent experiments. We identify CD28 as novel target antigen for pediatric T-ALL and provide evidence that CD28 is an immunotarget with functional relevance for T-ALL. We demonstrate the feasibility of CD28 CAR T cell generation and that these novel CAR T cells perform equally well as CD7 CAR T cells both in vitro and in vivo. Novel and functionally relevant CAR targets will facilitate clinical development of immunotherapy for T-lineage malignancies.
Resumen
Las luxofracturas hamate metacarpianas abarcan una amplia gama de lesiones complejas, raras en su aparición; suelen originarse al golpear con el puño objetos sólidos o por accidentes de alta ...energía. Clínicamente, el paciente presenta dolor y aumento de volumen en el borde cubital proximal de la mano. El diagnóstico definitivo esta dado por imágenes, en primera instancia, radiografías en cuatro proyecciones: postero anterior, oblicua en 30 grados, 60 grados y lateral, las cuales muestran el desplazamiento y rasgos de fractura. Idealmente debe complementarse con tomografía computada. El tratamiento es quirúrgico en general. Si se logra reducción óptima de forma cerrada se puede fijar percutáneo, pero con bajo umbral para realizar reducción abierta fijándose con agujas de Kirschner, tornillos o placa con el fin de disminuir el riesgo de mal unión, artrosis postraumática y dolor crónico. Presentamos 4 casos de pacientes con esta patología, con un seguimiento de 38 meses en promedio, que se sometieron a tratamiento quirúrgico en el mismo centro, con excelentes resultados funcionales reflejados en un DASH promedio de 9 puntos. Mostramos, además, una síntesis de la evidencia disponible, para guiar el manejo de una lesión de baja frecuencia, pero de importante implicancia para el pronóstico funcional de la mano.
Abstract
Metacarpal hamate fractures and dislocations comprise a wide range of rare, complex injuries. Their most common cause is hitting solid objects with the fist or sustaining high-energy traumas. The clinical picture consists of pain and swelling in the proximal ulnar side of the hand. Imaging, especially posteroanterior, oblique (30 and 60 degrees), and lateral radiographs, reveals fractures and dislocations, providing a definite diagnosis. Ideally, a computed tomography scan should complement imaging. Treatment is often surgical. Fixation may be percutaneous for optimal closed reduction, but with a low threshold for open reduction using Kirschner wires, screws, or plates to reduce the risk of malunion, post-traumatic osteoarthritis, and chronic pain. We present four patients with an average follow-up of 38 months who underwent surgical treatment in the same medical center, with excellent functional outcomes and mean Disabilities of the Arm, Shoulder, and Hand (DASH) score of 9 points. We also synthesized the available evidence to guide the management of a low-frequency injury with significant prognostic implications.
Hallux rigidus: Conceptos actuales Andrade, Paulina Alejandra Ferrada; Villanueva, Francisco Javier Sánchez; Hernández, Carlos Felipe Muñoz
Revista chilena de ortopedia y traumatologia,
04/2023, Letnik:
64, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Resumen
El hallux rigidus es una causa frecuente de consulta traumatológica, y es la artrosis más frecuente del pie. Se presenta clínicamente con dolor y pérdida de movimiento de la primera ...articulación metatarsofalángica, con impacto en la calidad de vida del paciente. El tratamiento comienza de forma conservadora, y tiene éxito en la mitad de los casos, con medidas, que incluyen modificación del calzado, manejo del peso y ortesis, diseñadas para limitar el movimiento de esta articulación. El tratamiento quirúrgico en pacientes con hallux rigidus leve a moderado se centra en eliminar los osteofitos dorsales con una queilectomía, que se puede asociar a una osteotomía de falange proximal o del primer metatarsiano, lo que mejora el rango de movimiento hacia un arco menos doloroso. En el hallux rigidus de alto grado, lo más aconsejado es la artrodesis, la cual proporciona alivio del dolor al costo de alterar la biomecánica de la marcha, dada la rigidez adquirida. Distintas artroplastias (resección, interposición, o protésicas) mejoran el dolor significativamente sin sacrificar movilidad, siendo una alternativa más fisiológica, que preserva la biomecánica del pie en pacientes seleccionados.
Abstract
Hallux rigidus is a frequent cause of traumatology consultations, and it is the most frequent osteoarthritis of the foot. It presents clinically with pain and loss of movement of the first metatarsophalangeal joint, with an impact on the quality of life of the patient. The treatment starts conservatively, and is successful in half of the cases, with measures such as modification of footwear, weight management and orthosis designed to limit the movement of the metatarsophalangeal joint. The surgical treatment in patients with mild to moderate hallux rigidus focuses on removing the dorsal osteophytes with a cheilectomy, which can be associated with a proximal phalanx or first metatarsal osteotomy, which improves the range of motion towards a less painful arch. In high-grade hallux rigidus, the most recommended treatment is still arthrodesis, which provides pain relief at the cost of altering gait biomechanics0 given the acquired stiffness. Different arthroplasties (resection, interposition or prosthetic) significantly improve pain without sacrificing mobility, and are a more physiological alternative to preserve the biomechanics of the foot in selected patients.
Evolución de la Cirugía de Epifisiodesis Sepúlveda O., Matías; Ferrada A., Paulina; Miranda-Chacón, Zaray ...
Andes pediatrica : revista Chilena de pediatría,
11/2021, Letnik:
92, Številka:
5
Journal Article
Recenzirano
Odprti dostop
La modulación quirúrgica del crecimiento fisiario, más conocida actualmente como “Crecimiento Guiado”, es un procedimiento utilizado como tratamiento para las diferencias de longitud o alteraciones ...de ejes de las extremidades. Si bien su uso se considera en aumento por el desarrollo de nuevas técnicas menos invasivas, no existen análisis estadísticos en la literatura que comprueben dicha afirmación.Objetivo: Evaluar la incidencia de la cirugía codificada como “Epifisiodesis (Fémur y/o Tibia)” en la población chilena desde el 1 de enero del 2010 al 31 de diciembre del 2019, los días de estadía hospitalaria relacionada con dicho código, y los datos demográficos de la población estudiada.Pacientes y Método: Se obtuvo desde la base de datos del Departamento de Estadísticas e Información de Salud (DEIS) de Chile, todos los egresos hospitalarios desde el 1 de enero del 2010 al 31 de diciembre del 2019, desde donde se extrajeron los resultados con el código de “Epifisiodesis (Fémur y/o Tibia)”, en menores de 15 años. Se analizó el número de procedimientos por año, número de días de estadía hospitalaria, edad y sexo de los pacientes.Resultados: El número de procedimientos codificados como “Epifisiodesis (Fémur y/o Tibia)” fue en aumento en Chile, durante el período analizado. Los días de estadía hospitalaria asociados a dicho procedimiento disminuyeron de forma progresiva entre los años 2012 y 2017. El procedimiento se realizó principalmente en mujeres, y en el grupo de 5-9 años, mostrando una tendencia al alza en niños del grupo de 10-14 años entre los años 2015 y 2019.Conclusiones: Se debe realizar mayores análisis para determinar los factores que producen estos resultados, los que probablemente se relacionen a acceso a técnicas más eficaces y sencillas, con menor morbilidad.
Evolución de la Cirugía de Epifisiodesis Sepúlveda O., Matías; Ferrada A., Paulina; Miranda-Chacón, Zaray ...
Andes pediatrica : revista Chilena de pediatría,
2021
Journal Article
Recenzirano
Odprti dostop
Resumen: Introducción: La modulación quirúrgica del crecimiento fisiario, más conocida actualmente como “Crecimiento Guiado”, es un procedimiento utilizado como tratamiento para las diferencias de ...longitud o altera ciones de ejes de las extremidades. Si bien su uso se considera en aumento por el desarrollo de nue vas técnicas menos invasivas, no existen análisis estadísticos en la literatura que comprueben dicha afirmación. Objetivo: Evaluar la incidencia de la cirugía codificada como “Epifisiodesis (Fémur y/o Tibia)” en la población chilena desde el 1 de enero del 2010 al 31 de diciembre del 2019, los días de estadía hospitalaria relacionada con dicho código, y los datos demográficos de la población estudiada. Pacientes y Método: Se obtuvo desde la base de datos del Departamento de Estadísticas e Informa ción de Salud (DEIS) de Chile, todos los egresos hospitalarios desde el 1 de enero del 2010 al 31 de diciembre del 2019, desde donde se extrajeron los resultados con el código de “Epifisiodesis (Fémur y/o Tibia)”, en menores de 15 años. Se analizó el número de procedimientos por año, número de días de estadía hospitalaria, edad y sexo de los pacientes. Resultados: El número de procedimientos codificados como “Epifisiodesis (Fémur y/o Tibia)” fue en aumento en Chile, durante el período analizado. Los días de estadía hospitalaria asociados a dicho procedimiento disminuyeron de forma progresiva entre los años 2012 y 2017. El procedimiento se realizó principalmente en mujeres, y en el grupo de 5-9 años, mostrando una tendencia al alza en niños del grupo de 10-14 años entre los años 2015 y 2019. Conclusiones: Se debe realizar mayores análisis para determinar los factores que produ cen estos resultados, los que probablemente se relacionen a acceso a técnicas más eficaces y sencillas, con menor morbilidad.
Objectives
Exploiting the forces of human T cells for treatment has led to the current paradigm of emerging immunotherapy strategies. Genetic engineering of the T-cell receptor (TCR) redirects ...specificity, ablates alloreactivity and brings significant progress and off-the-shelf options to emerging adoptive T-cell transfer (ACT) approaches. Targeted CRISPR/Cas9-mediated double-strand breaks in the DNA enable knockout or knock-in engineering.
Methods
Here, we perform CRISPR/Cas9-mediated TCR knockout using a therapeutically relevant ribonucleoprotein (RNP) delivery method to assess the safety of genetically engineered T-cell products. Whole-genome sequencing was performed to analyse whether CRISPR/Cas9-mediated DNA double-strand break at the TCR locus is associated with off-target events in human primary T cells.
Results
TCRα chain and TCRβ chain knockout leads to high on-target InDel frequency and functional knockout. None of the predicted off-target sites could be confirmed experimentally, whereas whole-genome sequencing and manual Integrative Genomics Viewer (IGV) review revealed 9 potential low-frequency off-target events genome-wide. Subsequent amplification and targeted deep sequencing in 7 of 7 evaluable loci did not confirm these low-frequency InDels. Therefore, off-target events are unlikely to be caused by the CRISPR/Cas9 engineering.
Conclusion
The combinatorial approach of whole-genome sequencing and targeted deep sequencing confirmed highly specific genetic engineering using CRISPR/Cas9-mediated TCR knockout without potentially harmful exonic off-target effects.
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