“Edición génica porcina para...

Xenotransplantation could be used to overcome the current shortage of organs for transplants. The technique consists of using animal (e.g. pig) organs to transplant them into \nhuman beings. Pigs are considered excellent candidates for xenotransplantation due to their anatomical and physiological si...

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Autor principal: Briski, Olinda
Otros Autores: Salamone, Daniel Felipe
Formato: Tesis doctoral acceptedVersion
Lenguaje:Español
Publicado: Universidad de Buenos Aires. Facultad de Ciencias Veterinarias 2023
Materias:
Acceso en línea:http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_7379
https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_7379.dir/7379.PDF
Aporte de:
id I28-R145-HWA_7379
record_format dspace
institution Universidad de Buenos Aires
institution_str I-28
repository_str R-145
collection Repositorio Digital de la Universidad de Buenos Aires (UBA)
language Español
orig_language_str_mv spa
topic CRISPR-cas9
Embriones
Cerdos
CRISPR-cas9
Embryos
Pigs
Espermatozoide
Porcinos
Xenotrasnplante
Ciencias Veterinarias
spellingShingle CRISPR-cas9
Embriones
Cerdos
CRISPR-cas9
Embryos
Pigs
Espermatozoide
Porcinos
Xenotrasnplante
Ciencias Veterinarias
Briski, Olinda
“Edición génica porcina para...
topic_facet CRISPR-cas9
Embriones
Cerdos
CRISPR-cas9
Embryos
Pigs
Espermatozoide
Porcinos
Xenotrasnplante
Ciencias Veterinarias
description Xenotransplantation could be used to overcome the current shortage of organs for transplants. The technique consists of using animal (e.g. pig) organs to transplant them into \nhuman beings. Pigs are considered excellent candidates for xenotransplantation due to their anatomical and physiological similarities with the human species, as well as the facility to reproduce and keep them under appropriate biosecurity conditions. However, multiple genetic \nmodifications of the pig genome will be necessary to reduce immunologic organ rejection in order for xenotransplantation to be viable. The development of the CRISPR-Cas9 system has \nenabled testing genetic modifications through various embryo production techniques, such as \nsomatic cell nuclear transfer, oocyte or zygote microinjection, or electroporation derived from \neither in vitro fertilization (IVF) or in vivo fertilization. In this thesis, we propose the use of intracytoplasmic sperm injection mediated gene editing (ICSI-MGE) to produce genetically \nedited embryos, which has not been a broadly used alternative so far. ICSI-MGE consists in the co-injection of the spermatozoon together with the CRISPR-Cas9 system in a mature oocyte. ICSI-MGE was used in this thesis to inactivate certain genes that encode enzymes \nresponsible for the hyperacute immunological rejection generated during pig-to-human \nxenotransplantation, such as alpha-1,3-galactosyltransferase (GGTA1), citidine \nmonophosphate N-acetylneuraminic acid hydrolase (CMAH) and beta-1,4-N-acetyl galactosaminil transferase 2 (4GalNT2). Concurrently, we sought to inactivate the gene that codes for the growth hormone receptor (GHR) in order to limit the growth of the pig for its organs to be similar in size to those of the human being. This is an indispensable requirement for future heart transplants. \nThe initial experiments focused on optimizing porcine ICSI and exploring its biological limitations. Porcine ICSI has the advantage of avoiding polyspermy, which is very frequent in porcine IVF. However, the blastocyst rates obtained were still low (8.4 to 22%), which could be related to various causes, such as incomplete sperm decondensation and/or alteration in the oocyte activation mechanism. Consequently, as initial measures we proposed to improve \nthe two-pronucleus formation rates (2-PN) by using the piezo-drill (ICSIp), the electrically \nassisted activation (ICSIe) and the chemically assisted activation with the zinc chelator 1,10-\nphenantroline (PHEN) (ICSI-phen). The results showed that neither ICSIe, ICSIp or ICSI-phen improved the 2-PN formation rates compared to conventional ICSI (ICSI). ICSI-phen resulted \nin higher activation rates than conventional ICSI (82.5% and 55.88%, respectively), but it did \nnot contribute to the decondensation of the spermatozoon. Subsequently, we evaluated the embryo development to blastocyst comparing ICSI with ICSI-phen and no significant \ndifferences were found in the blastocyst rates (ICSI=13.85%; ICSI-phen=20.5%) or in their \nquality after evaluating the expression pattern of pluripotency markers. This demonstrated that, \nwhile PHEN does not produce improvements, it does not have negative effects on the amount \nor quality of the embryo either. In summary, the proposed strategies to improve the efficiency \nof porcine ICSI do not increase the 2-PN formation rates, nor the blastocyst rates or their \nquality. In addition, artificial assisted activation showed certain risk of parthenogenetic embryo \nproduction, and its use was therefore not considered in future experiments in this thesis. \nThe final goal of the thesis was to use ICSI-MGE to produce genetically edited pigs and compare embryo developmental rates, embryo gene editing rates, and birth rates of edited \npiglets with those resulting from two other techniques: i) oocyte microinjection followed by IVF,\nand ii) zygote microinjection produced by in vivo fertilization with CRISPR-Cas9 system. Our \nresults showed that ICSI-MGE was efficient in editing the GGTA1 gene, as 100% of the embryos were edited (15/15), comparable to the efficiency registered by microinjection of the \noocyte and subsequent IVF (16/16). The viability of the embryos fertilized in vivo and microinjected with CRISPR-Cas9 was higher than for the other two experimental groups: it was the only group from which five offspring were born following the surgical transfer of the embryos to a recipient female. The five born piglets presented editions in both alleles of the GGTA1 gene. Furthermore, three of these piglets also presented an edited GHR gene, one biallelic and the other two monoallelic. No effective editions for the CMAH and 4GalNT2 \ngenes were observed neither in the blastocysts obtained (regardless of which of the three\ntechniques is used) nor in the born piglets. \nIn conclusion, it is possible to generate blastocysts with high mutation rates through \nICSI-MGE. This technique is presented as an alternative and a solution to the polyspermy resulting from porcine IVF. Additionally, ICSI-MGE can be a very useful solution in various scenarios, such as a limited availability of male samples of high genetic value (and/or if they are genetically modified), as well as in cases in which semen shows poor response to cryopreservation or poor sperm quality and is thus unable to achieve fertilization through IVF \nor artificial insemination. However, although ICSI-MGE mutation rates are higher than those obtained by microinjection of embryos fertilized in vivo, the viability of embryos produced in vitro is yet to improve by standardizing culture media for porcine, which is an important step for the viable production of genetically edited pigs. Obtaining embryos fertilized in vivo requires \ngreater logistics and effort when compared to the production of embryos with in vitro techniques, and the mutation rates are lower as well. However, it was the technique that proved \nefficient for the birth of five edited pigs. While more tests will be required to obtain all of the \nproposed modifications simultaneously, they are the first genetically edited pigs achieved in Latin America and contribute a significant advance in pig production for xenotransplantation
author2 Salamone, Daniel Felipe
author_facet Salamone, Daniel Felipe
Briski, Olinda
format Tesis doctoral
Tesis doctoral
acceptedVersion
author Briski, Olinda
author_sort Briski, Olinda
title “Edición génica porcina para...
title_short “Edición génica porcina para...
title_full “Edición génica porcina para...
title_fullStr “Edición génica porcina para...
title_full_unstemmed “Edición génica porcina para...
title_sort “edición génica porcina para...
publisher Universidad de Buenos Aires. Facultad de Ciencias Veterinarias
publishDate 2023
url http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_7379
https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_7379.dir/7379.PDF
work_keys_str_mv AT briskiolinda ediciongenicaporcinapara
AT briskiolinda ediciongenicaporcinaparaxenotrasplantemediadaporlainyeccionintracitoplasmaticadeespermatozoide
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spelling I28-R145-HWA_73792024-08-27 “Edición génica porcina para... Xenotransplantation could be used to overcome the current shortage of organs for transplants. The technique consists of using animal (e.g. pig) organs to transplant them into \nhuman beings. Pigs are considered excellent candidates for xenotransplantation due to their anatomical and physiological similarities with the human species, as well as the facility to reproduce and keep them under appropriate biosecurity conditions. However, multiple genetic \nmodifications of the pig genome will be necessary to reduce immunologic organ rejection in order for xenotransplantation to be viable. The development of the CRISPR-Cas9 system has \nenabled testing genetic modifications through various embryo production techniques, such as \nsomatic cell nuclear transfer, oocyte or zygote microinjection, or electroporation derived from \neither in vitro fertilization (IVF) or in vivo fertilization. In this thesis, we propose the use of intracytoplasmic sperm injection mediated gene editing (ICSI-MGE) to produce genetically \nedited embryos, which has not been a broadly used alternative so far. ICSI-MGE consists in the co-injection of the spermatozoon together with the CRISPR-Cas9 system in a mature oocyte. ICSI-MGE was used in this thesis to inactivate certain genes that encode enzymes \nresponsible for the hyperacute immunological rejection generated during pig-to-human \nxenotransplantation, such as alpha-1,3-galactosyltransferase (GGTA1), citidine \nmonophosphate N-acetylneuraminic acid hydrolase (CMAH) and beta-1,4-N-acetyl galactosaminil transferase 2 (4GalNT2). Concurrently, we sought to inactivate the gene that codes for the growth hormone receptor (GHR) in order to limit the growth of the pig for its organs to be similar in size to those of the human being. This is an indispensable requirement for future heart transplants. \nThe initial experiments focused on optimizing porcine ICSI and exploring its biological limitations. Porcine ICSI has the advantage of avoiding polyspermy, which is very frequent in porcine IVF. However, the blastocyst rates obtained were still low (8.4 to 22%), which could be related to various causes, such as incomplete sperm decondensation and/or alteration in the oocyte activation mechanism. Consequently, as initial measures we proposed to improve \nthe two-pronucleus formation rates (2-PN) by using the piezo-drill (ICSIp), the electrically \nassisted activation (ICSIe) and the chemically assisted activation with the zinc chelator 1,10-\nphenantroline (PHEN) (ICSI-phen). The results showed that neither ICSIe, ICSIp or ICSI-phen improved the 2-PN formation rates compared to conventional ICSI (ICSI). ICSI-phen resulted \nin higher activation rates than conventional ICSI (82.5% and 55.88%, respectively), but it did \nnot contribute to the decondensation of the spermatozoon. Subsequently, we evaluated the embryo development to blastocyst comparing ICSI with ICSI-phen and no significant \ndifferences were found in the blastocyst rates (ICSI=13.85%; ICSI-phen=20.5%) or in their \nquality after evaluating the expression pattern of pluripotency markers. This demonstrated that, \nwhile PHEN does not produce improvements, it does not have negative effects on the amount \nor quality of the embryo either. In summary, the proposed strategies to improve the efficiency \nof porcine ICSI do not increase the 2-PN formation rates, nor the blastocyst rates or their \nquality. In addition, artificial assisted activation showed certain risk of parthenogenetic embryo \nproduction, and its use was therefore not considered in future experiments in this thesis. \nThe final goal of the thesis was to use ICSI-MGE to produce genetically edited pigs and compare embryo developmental rates, embryo gene editing rates, and birth rates of edited \npiglets with those resulting from two other techniques: i) oocyte microinjection followed by IVF,\nand ii) zygote microinjection produced by in vivo fertilization with CRISPR-Cas9 system. Our \nresults showed that ICSI-MGE was efficient in editing the GGTA1 gene, as 100% of the embryos were edited (15/15), comparable to the efficiency registered by microinjection of the \noocyte and subsequent IVF (16/16). The viability of the embryos fertilized in vivo and microinjected with CRISPR-Cas9 was higher than for the other two experimental groups: it was the only group from which five offspring were born following the surgical transfer of the embryos to a recipient female. The five born piglets presented editions in both alleles of the GGTA1 gene. Furthermore, three of these piglets also presented an edited GHR gene, one biallelic and the other two monoallelic. No effective editions for the CMAH and 4GalNT2 \ngenes were observed neither in the blastocysts obtained (regardless of which of the three\ntechniques is used) nor in the born piglets. \nIn conclusion, it is possible to generate blastocysts with high mutation rates through \nICSI-MGE. This technique is presented as an alternative and a solution to the polyspermy resulting from porcine IVF. Additionally, ICSI-MGE can be a very useful solution in various scenarios, such as a limited availability of male samples of high genetic value (and/or if they are genetically modified), as well as in cases in which semen shows poor response to cryopreservation or poor sperm quality and is thus unable to achieve fertilization through IVF \nor artificial insemination. However, although ICSI-MGE mutation rates are higher than those obtained by microinjection of embryos fertilized in vivo, the viability of embryos produced in vitro is yet to improve by standardizing culture media for porcine, which is an important step for the viable production of genetically edited pigs. Obtaining embryos fertilized in vivo requires \ngreater logistics and effort when compared to the production of embryos with in vitro techniques, and the mutation rates are lower as well. However, it was the technique that proved \nefficient for the birth of five edited pigs. While more tests will be required to obtain all of the \nproposed modifications simultaneously, they are the first genetically edited pigs achieved in Latin America and contribute a significant advance in pig production for xenotransplantation Fil: Briski, Olinda. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias Salamone, Daniel Felipe Briski, Olinda 2023-10-11 El xenotrasplante es una opción para superar la escasez de órganos para trasplantes. \nConsiste en utilizar órganos de animales, como cerdos, para trasplantarlos a seres humanos. \nEl cerdo es considerado una excelente opción debido a las similitudes anatómicas y \nfisiológicas con la especie humana, así como las facilidades para reproducirlo y mantenerlo \nen condiciones de bioseguridad. Sin embargo, antes de que el xenotrasplante pueda ser una realidad clínica, será necesario realizar múltiples modificaciones genéticas del genoma porcino que permitan reducir el rechazo inmunológico del órgano. Con el desarrollo del sistema CRISPR-Cas9 se han ensayado diversas técnicas de producción de embriones para \nintroducir modificaciones genéticas, como la clonación a partir de células somáticas, la \nmicroinyección o electroporación de ovocitos o de cigotos derivados de fecundación in vitro\n(FIV) o de fecundación in vivo. Una alternativa menos explorada para producir embriones \ngenéticamente editados y que proponemos en esta tesis es la edición génica mediada por la \ninyección intracitoplasmática del espermatozoide (ICSI-MGE). La misma consiste en la coinyección del espermatozoide junto al sistema CRISPR-Cas9 en un ovocito maduro. En \nesta tesis este sistema se utilizó para inactivar determinados genes que codifican para enzimas responsables del rechazo inmunológico hiperagudo generados durante el \nxenotrasplante de cerdo a humanos como son la alpha-1,3 galactosyltransferasa (GGTA1), la hidrolasa del ácido N-acetilneuramínico citidina monofosfato (CMAH) y beta-1,4-N-acetil \ngalactosaminil transferasa 2 (?4GalNT2). A la vez, también, se buscó inactivar el gen que codifica para el receptor de la hormona de crecimiento (GHR) limitando el crecimiento del cerdo para que sus órganos sean similar en tamaño a los del ser humano, requisito \nindispensable para futuros trasplantes de corazón.\nLos primeros experimentos de la tesis se enfocaron en optimizar la ICSI porcina y \nexplorar sus limitaciones biológicas. La ICSI en porcinos tiene la ventaja de evitar la polispermia que es muy frecuente en la FIV porcina. Sin embargo, las tasas de blastocistos obtenidas aún son reducidas (8,4 a 22%) y esto podría relacionarse con diversas causas, como a) la descondensación incompleta del espermatozoide y/o, b) alteración en el \nmecanismo de activación del ovocito. Por lo tanto, en primer lugar, propusimos mejorar las tasas de formación de dos pronúcleos (2-PN) mediante la utilización del piezo-drill (ICSIp), la activación asistida eléctricamente (ICSIe) y la activación asistida químicamente con el quelante de zinc 1,10-fenantrolina (PHEN) (ICSI-phen). Los resultados demostraron que ni la \nICSIe, ICSIp o el ICSI-phen mejoraron las tasas de formación de 2-PN respecto a la ICSI convencional (ICSI). La ICSI-phen resultó en tasas de activación superiores a la ICSI \nconvencional (82,5% y 55,88 %, respectivamente), sin embargo, no contribuyó en la descondensación del espermatozoide. Posteriormente, evaluamos el desarrollo embrionario \na blastocisto comparando la ICSI con la ICSI-phen y no se hallaron diferencias significativas en las tasas de blastocistos (ICSI=13,85%; ICSI-phen=20,5%), ni en la calidad de los mismos \nluego de evaluar el patrón de expresión de marcadores de pluripotencia. Con esto \ndemostramos que, si bien el PHEN no produce mejoras, tampoco tiene efectos negativos en la cantidad o la calidad embrionaria. En resumen, las estrategias propuestas para mejorar la eficiencia de la ICSI porcina no incrementan las tasas de formación de 2-PN, ni las tasas de blastocisto o la calidad de estos. Además, la activación asistida artificial demostró cierto riesgo \nde producir embriones partenogénicos, por lo que su utilización no fue considerada en futuros \nexperimentos de esta tesis. \nEl último objetivo de la tesis fue utilizar la ICSI-MGE para producir cerdos \ngenéticamente editados y comparar las tasas de desarrollo embrionario, las tasas de edición\ngénica en embriones y las tasas de nacimientos de animales editados con las técnicas de: i) \nmicroinyección de ovocitos seguidos de FIV, y ii) microinyección de cigotos producidos por \nfecundación in vivo con el sistema CRISPR-Cas9. Nuestros resultados demostraron que la \nICSI-MGE resultó eficiente en la edición del gen GGTA1 obteniendo un 100% de embriones editados (15/15), comparables a aquellos obtenidos por microinyección del ovocito y posterior FIV (16/16). La viabilidad de los embriones fecundados in vivo y microinyectados con \nCRISPR-Cas9 resultó ser la más alta comparada con los otros dos grupos experimentales, siendo el único grupo del cuál se lograron obtener crías nacidas luego de transferir los embriones quirúrgicamente a una hembra receptora. Los cinco lechones nacidos presentaron ediciones en los dos alelos del gen GGTA1. Además, tres de estos lechones también tienen editados el gen GHR, uno bialélico y los otros dos monoalélicos. No se obtuvieron ediciones \nefectivas para los genes CMAH y 4GalNT2 en los blastocistos obtenidos por ninguna de las\ntres técnicas, ni en los animales nacidos.\nEn conclusión, es posible generar blastocistos con altas tasas de mutación mediante la ICSI-MGE. Esta técnica se presenta como una alternativa y solución a la polispermia presente en la FIV porcina. Además, puede resultar de gran utilidad en diversas situaciones como la disponibilidad limitada de muestras de machos de alto valor genético y/o que estén \ngenéticamente modificados, así como en casos de semen con mala respuesta a la criopreservación o mala calidad espermática e incapaz de fecundar por FIV o inseminación artificial. Sin embargo, si bien las tasas de mutación de ICSI-MGE son más altas que las obtenidas pormicroinyección de cigotos fecundados in vivo, aún se debe continuar mejorando \nla viabilidad de los embriones producidos in vitro mediante la estandarización de medios de cultivo para porcinos, un paso importante para la producción viable de cerdos genéticamente editados. La obtención de cigotos fecundados in vivo requiere de una mayor logística y trabajo \ny las tasas de mutación son menores comparadas con las obtenidas en la producción de cigotos con técnicas in vitro. Sin embargo, fue la técnica que resultó eficiente para el \nnacimiento de cinco cerdos editados. Si bien se requerirán de más ensayos para obtener en \nsimultáneo todas las modificaciones propuestas, se obtuvieron los primeros cerdos genéticamente editados logrados en Latinoamérica y contribuyen a un avance en la producción de cerdos para xenotrasplante. application/pdf CRISPR-cas9 Embriones Cerdos CRISPR-cas9 Embryos Pigs spa Universidad de Buenos Aires. Facultad de Ciencias Veterinarias info:eu-repo/semantics/openAccess htpps://creativecommons.org/licenses/by-nend/2.5/ar/ Espermatozoide Porcinos Xenotrasnplante Ciencias Veterinarias Doctora de Universidad de Buenos Aires en Ciencias Veterinarias Edición génica porcina para xenotrasplante mediada por la inyección intracitoplasmática de espermatozoide info:eu-repo/semantics/doctoralThesis info:ar-repo/semantics/tesis doctoral info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_7379 https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_7379.dir/7379.PDF