Littérature scientifique sur le sujet « Gene editing germinale »

Dovremmo accogliere tutti i possibili sviluppi dell’editing genetico? L’editing genetico delle cellule somatiche potrebbe essere considerato alla pari delle terapie convenzionali volte a trattare particolari patologie o ad alleviarne i sintomi. Tale intervento interesserebbe esclusivamente il singolo paziente trattato. Esso potrebbe quindi essere ben accolto come un nuovo tipo di trattamento per i tumori e le malattie del sangue, come ad esempio la beta-talassemia. Diversamente, l’editing della linea germinale avrebbe effetti ereditari. Ciò solleva preoccupazioni particolari riguardo al rischio medico. I rischi medici non sono, tuttavia, gli unici tipi di rischi che possono derivare dalla modificazione genetica della linea germinale. Nel contributo non vengono discussi i rischi medici, ma quelli sociali e morali correlati alla manipolazione genetica della linea-germinale. ---------- Should we welcome all developments in gene editing? Somatic cell gene editing would be on a par with conventional therapies aimed at treating particular conditions or alleviating symptoms. It would solely affect the individual patient treated. It could thus serve as a welcome new kind of treatment for cancers and blood diseases such as ß-thalassaemia. Germ-line gene editing, on the other hand, would have hereditary effects. This raises special concerns about medical mishaps. Medical risks are, however, not the only kinds of risks in the case of germline gene editing. Discussed here are not the medical risks, but the social and moral risks of germ-line-gene editing.

Homologous recombination (HR) in somatic cells is not as well understood as meiotic recombination and is thought to be rare. In a previous study, we showed that Inter-Homologous Somatic Recombination (IHSR) can be achieved by targeted induction of DNA double-strand breaks (DSBs). Here, we designed a novel IHSR assay to investigate this phenomenon in greater depth. We utilized F1 hybrids from divergent parental lines, each with a different mutation at the Carotenoid isomerase (CRTISO) locus. IHSR events, namely crossover or gene conversion (GC), between the two CRTISO mutant alleles (tangerine color) can restore gene activity and be visualized as gain-of-function, wildtype (red) phenotypes. Our results show that out of four intron DSB targets tested, three showed DSB formation, as seen from non-homologous end-joining (NHEJ) footprints, but only one target generated putative IHSR events as seen by red sectors on tangerine fruits. F2 seeds were grown to test for germinal transmission of HR events. Two out of five F1 plants showing red sectors had their IHSR events germinally transmitted to F2, mainly as gene conversion. Six independent recombinant alleles were characterized: three had truncated conversion tracts with an average length of ~1 kb. Two alleles were formed by a crossover as determined by genotyping and characterized by whole genome sequencing. We discuss how IHSR can be used for future research and for the development of novel gene editing and precise breeding tools.

Most Hieracium subgenus Pilosella species are self-incompatible. Some undergo facultative apomixis where most seeds form asexually with a maternal genotype. Most embryo sacs develop by mitosis, without meiosis and seeds form without fertilization. Apomixis is controlled by dominant loci where recombination is suppressed. Loci deletion by γ-irradiation results in reversion to sexual reproduction. Targeted mutagenesis of genes at identified loci would facilitate causal gene identification. In this study, the efficacy of CRISPR/Cas9 editing was examined in apomictic Hieracium by targeting mutations in the endogenous PHYTOENE DESATURASE (PDS) gene using Agrobacterium-mediated leaf disk transformation. In three experiments, the expected albino dwarf-lethal phenotype, characteristic of PDS knockout, was evident in 11% of T0 plants, 31.4% were sectorial albino chimeras, and the remainder were green. The chimeric plants flowered. Germinated T1 seeds derived from apomictic reproduction in two chimeric plants were phenotyped and sequenced to identify PDS gene edits. Up to 86% of seeds produced albino seedlings with complete PDS knockout. This was attributed to continuing Cas9-mediated editing in chimeric plants during apomictic seed formation preventing Cas9 segregation from the PDS target. This successful demonstration of efficient CRISPR/Cas9 gene editing in apomictic Hieracium, enabled development of the discussed strategies for future identification of causal apomixis genes.

Abstract Receptor editing, clonal deletion and anergy are the key components of the central tolerance. It is well characterized that receptor editing plays the most important role among the three mechanisms. We recently identified a post germinal center tolerance check point, where receptor editing is re-induced to diminish the autoreactive B cells generated by class switching and somatic hypermutation. Here we demonstrate that this de-novo re-induction of the recombinase RAG gene in antigen-activated B cells is reduced by neutralization of IL-6. Serum level of autoreactive anti-dsDNA antibodies is increased in mice immunized with a peptide mimetope of dsDNA if they are given antibodies to IL-6 to reduce receptor editing. The same effect is observed in mice haploid for IL-6 when compared to WT mice. This dependence on IL-6 is B cell intrinsic, as demonstrated in adoptive transfer experiments when B cells from either WT mice or IL-6+/- mice were reconstituted in uMT mice. Our data reveal a novel but crucial role of IL-6 in regulation of the B cell autoimmunity, and show that Il-6 serves a regulatory function as well as being proinflammatory.

Mouse germinal center (GC) B cells have been shown to undergo secondary V(D)J (V, variable; D, diversity; J, joining) recombination (receptor editing) mediated by the reexpressed products of recombination activating gene (RAG)-1 and RAG-2. We show here that interleukin (IL)-7 as well as IL-4 was effective in inducing functional RAG products in mouse IgD+ B cells activated via CD40 in vitro. Blocking of the IL-7 receptor (IL-7R) by injecting an anti– IL-7R monoclonal antibody resulted in a marked suppression of the reexpression of RAG-2 and subsequent V(D)J recombination in the draining lymph node of immunized mice, whereas RAG-2 expression was not impaired in immunized IL-4–deficient mice. Further, these peripheral B cells activated in vitro or in vivo were found to express IL-7R. These findings indicate a novel role for IL-7 and IL-7R in inducing receptor editing in GC B cells.

V(D)J (V, variable; D, diversity; J, joining) combination of immunoglobulin (Ig) genes established in premature B cells has been thought to be conserved throughout differentiation at mature stages. However, germinal center (GC) B cells have been shown to reexpress recombination-activating gene (RAG)-1 and RAG-2 proteins in immunized mice. Here, we present several lines of evidence indicating that RAG proteins thus induced are functional as the V(D)J recombinase. DNA excision product reflecting Vλ1 to Jλ1 rearrangement was generated in parallel with the expression of RAG genes in mature mouse B cells that were activated in vitro with LPS and IL-4. Similar λ chain gene rearrangement was observed in the draining lymph node of immunized mice. Further, B cells that underwent λ gene rearrangement were shown by in situ PCR to be localized within GCs. Thus, secondary rearrangement of Ig genes (receptor editing) can occur in mature B cells.

Abstract Hyper-IgM Type 1 (HIGM1) is caused by mutations of CD40L, whose absence in CD4 T cells impairs signaling for B cell activation and Ig class-switching. Since unregulated CD40L expression leads to lymphoproliferations/lymphomas in the mouse model of the disease, gene correction must preserve the physiological regulation of the gene. Gene editing of either autologous T cells or hematopoietic stem cells (HSC) held promise for treating HIGM1. We developed a "one size fits all" editing strategy to insert a 5'-truncated corrective CD40L cDNA in the first intron of the native human gene, effectively making expression conditional to targeted insertion in the intended locus. By exploiting a protocol that preserves T stem memory cells (TSCM), we reproducibly obtained ~40% of editing efficiency in healthy donor and patients derived T cells, restoring regulated, although partial, CD40L surface expression. The reconstituted level of expression, however, was sufficient to fully restore helper function to B cells. In order to select, track and potentially deplete edited T cells, we coupled the corrective cDNA with a clinically compatible selector gene and confirmed that enriched T cells preserved their engraftment capacity in NSG mice. Unexpectedly, the presence of an IRES-linked downstream coding frame counteracted the shorter half-life of transcript from the edited locus, allowing replenishment of intracellular stores and surface translocation of physiological amounts of CD40L upon activation. We also tailored the CD40L editing strategy to human HSC, reaching up to 15-30% editing in HSC long term engrafting NSG mice, depending on the HSC source. We then modelled the therapeutic potential of both T cell and HSC gene therapy by infusing increasing proportions of WT murine cells, as surrogates of edited cells, in HIGM1 mice. Administration of functional T cells at clinically relevant doses in HIGM1 mice, preconditioned or not with different lymphodepleting regimens, achieved long term stable T cell engraftment and partial rescue of antigen specific IgG response and germinal center formation in splenic follicles after vaccination with a thymus dependent antigen. Remarkably, infusion of T cells from mice pre-exposed to the antigen, mimicking treatment of chronically infected patients, was effective even in absence of conditioning and protected the mice from a disease relevant infection induced by the opportunistic pathogen Pneumocystis murina. Transplantation of functional T cells admixed with an equal number of HIGM1 T cells resulted in lower vaccination response, indicating competition between WT and HIGM1 cells and implying that increasing the fraction of corrected cells in the graft by selection would improve immune reconstitution. Concerning HSC gene therapy, transplanting 25% WT cells along with HIGM1 ones in HIGM1 mice - mirroring the editing efficiencies achieved in human HSC - rescued antigen specific IgG response and established protection from pathogen comparably to T cell therapy. These findings suggest that autologous edited T cells can provide immediate and substantial benefits to HIGM1 patients and position T cell as competitive strategy to HSC gene therapy, because of more straightforward translation, lower safety challenges and potentially comparable clinical benefits. We thus embarked in assessing GMP compliant reagents and protocols for T cell activation, culture and editing and developed a scalable manufacturing process. Optimization of clinical grade culture conditions allowed further increasing editing efficiency, total cellular yield and maintenance of TSCM thus paving the way to the design of a clinical trial. Disclosures Naldini: Genenta Science: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Abstract Class switching from IgM to IgG and IgA is essential for antiviral immunity and requires activation-induced cytidine deaminase (AID), an APOBEC family member with DNA-editing activity. Germinal center (GC) B cells express AID upon activation by CD4+ T cells through CD40 ligand (CD40L) and IL-4. HIV-1 is thought to impair IgG and IgA responses to viral antigens, opportunistic pathogens and vaccines by causing progressive loss of CD4+ T cells and by rendering B cells poorly responsive to CD4+ T cell help. It remains unknown whether HIV-1 targets AID to hamper protective IgG and IgA responses. We found that infected GCs contained less AID, but normal APOBEC3G, an AID-related RNA-editing protein. AID down-regulation was not associated with local loss of CD4+ T cells and CD40L, but rather correlated with decreased activation of AID-inducing transcription factors, such as NF-κB and STAT6, and with increased expression of feedback inhibitors of NF-κB and STAT6, including IκBα, SOCS1 and SOCS3. AID down-regulation also correlated with accumulation of the viral protein Nef in the GC and with trafficking of Nef within membrane channels connecting infected myeloid cells to B cells. Together with our recent in vitro studies showing that Nef penetrates B cells and inhibits class, the present in vivo data suggest that HIV-1 evades protective IgG and IgA responses by targeting the class switch recombinase machinery.

Abstract We examined the chronic lymphocytic leukemia (CLL) cells of 2457 patients evaluated by the CLL Research Consortium (CRC) and found that 63 (2.6%) expressed immunoglobulin (Ig) encoded by the Ig heavy-chain-variable-region gene (IGHV), IGHV3-21. We identified the amino acid sequence DANGMDV (motif-1) or DPSFYSSSWTLFDY (motif-2) in the Ig heavy-chain (IgH) third complementarity-determining region (HCDR3) of IgH, respectively, used by 25 or 3 cases. The IgH with HCDR3 motif-1 or motif-2, respectively, was paired with Ig light chains (IgL) encoded by IGLV3-21 or IGKV3-20, suggesting that these Ig had been selected for binding to conventional antigen(s). Cases that had HCDR3 motif-1 had a median time from diagnosis to initial therapy comparable with that of cases without a defined HCDR3 motif, as did cases that used mutated IGHV3-21 (n = 27) versus unmutated IGHV3-21 (n = 30). Of 7 examined cases that used Ig encoded by IGHV3-21/IGLV3-21, we found that 5 had a functionally rearranged IGKV allele that apparently had incurred antigendriven somatic mutations and subsequent rearrangement with KDE. This study reveals that CLL cells expressing IGHV3-21/IGLV3-21 most likely were derived from B cells that had experienced somatic mutation and germinal-center maturation in an apparent antigen-driven immune response before undergoing Ig-receptor editing and after germinal-center leukemogenic selection.

The CRISPR/Cas9 system has been widely utilized in plant biotechnology as a gene editing tool. However, a conventional design with ubiquitously expressed CRISPR/Cas9 was observed to cause large numbers of somatic mutations that complicated the identification of heritable mutations. We constructed a pollen-specific CRISPR/Cas9 (PSC) system using pollen-specific promoters of maize Profilin 1 and Profilin 3 (pZmPRO1 and pZmPRO3) to drive Cas9 expression, and the bZIP transcription factor Opaque2 (O2) was employed as the target gene. The maize ubiquitin promoter (pZmUbi)-driven CRISPR/Cas9 (UC) system was employed as a control. We generated transgenic plants for the PSC and UC systems and analyzed three independent events for each system. We found that the pZmPRO1 PSC system generated no target gene mutations in the T0 generation but successfully generated 0–90% target gene mutations in the T1 generation. A total of 31 of 33 mutations in the T1 generation could be inherited in the T2 generation. In addition, 88.9–97.3% of T2 mutations were from the T1 generation. The UC system generated mutations in the T0 generation, and 0%, 50% and 92.9% of T1 mutations were from the T0 generation. Our results demonstrate that the PSC system provided stable, heritable mutants in the next generation, and this approach might also be applied in other crops using germinal cell-specific CRISPR/Cas9 systems to facilitate plant breeding.

The dissertation aims at identifying and analyzing the scientific, legal, and ethical issues raised by the perspective of intentional modification of human germline by the potential future use of gene editing techniques in the context of human reproduction. Such a study makes it possible to formulate some critical considerations about human germline gene editing governance. The dissertation claims that the best option to regulate the use of this biotechnological innovation for reproductive purposes consists of a regulation on a state-by-state-basis, which should however be developed within an international governance framework. Several arguments are suggested to underpin this thesis, and some recent initiatives adhering to such governance pattern are examined. The research is organized in three chapters. The first chapter, which is introductive to the real research, focuses on the scientific and technical aspects of the thesis topic. More specifically, this chapter aims at laying the foundations for the subsequent discussion, by defining and explaining the notions of i) DNA, gene, chromosome; ii) genetic mutation and genetic disease; and iii) gene therapy and gene editing. Special attention is paid to this latter technology and especially to its potential use on the human germline. Such use is highly controversial, mainly – but not exclusively – since, unlike modifications made by somatic gene editing, those affecting germinal cells – namely, gametes and zygotes – are transmitted to descendants, and thus to next generations. The second chapter is divided into two sections. The first section reconstructs and analyses the existing regulations in the field of human germline gene editing at international, supranational and national level, stressing their vagueness, fragmentation and lack of specificity. Given the impossibility of extensively examining all relevant domestic laws, guidelines and policies, those of four countries only – the USA, the UK, China and Italy – have been considered in detail. This choice is motivated by the geographical and cultural representativeness of their respective regulations, as well as by the fact that, except for Italy, those countries conducted nearly all the experiments carried out so far in the field of human germline gene editing. The second section of the chapter precisely focuses on these experiments – both for research and reproductive purposes. Jiankui He’s experiment – which resulted in the birth of the world’s first gene-edited babies in 2018 – and Denis Rebrikov’s germline gene editing clinical trial project are thoroughly described and analyzed. The third and last chapter deals with the ethical issues raised by the perspective of the potential future implementation of germline gene editing interventions in the context of human reproduction. This chapter too is articulated into two sections. The first section provides the theoretical bases for the subsequent ethical analysis, by dividing the possible future uses of germline gene editing techniques into three categories: i) therapeutic interventions; ii) medical enhancement interventions; and iii) non-medical enhancement interventions. Such categorization is paramount, since the various ethical issues related to human germline gene editing do not always involve all three of these categories, and, even when they do, they tend to carry different connotations according to each category. This becomes clear in the second section of the chapter, which critically explores six main ethically problematic areas related to this biotechnological innovation and their numerous articulations. Finally, the dissertation argues that the scientific, legal and ethical issues identified and examined throughout the research must be taken into account by proper germline gene editing governance mechanisms, which should be the result of parallel and complementary regulatory initiatives promoted both at national and international level.

Il presente lavoro, incominciato nel novembre del 2017, è partito con l'ambizione di ricostruire la risposta che il sistema giuridico fornisce innanzi alle nuove tecniche di ingegneria genetica che, a fronte della loro applicabilità sugli esseri umani, hanno prodotto, negli ultimi anni, il sorgere di nuovi stakeholders e, ancor prima, di nuovi interessi meritevoli di tutela. Se fino a qualche anno fa pareva impensabile modificare il genoma umano e, men che meno, farlo in maniera precisa, efficiente ed economica, oggi grazie al sistema di modificazione genetica CRISPR/Cas9 è possibile, intervenendo sulla linea germinale degli embrioni umani, prevenire la contrazione di odiose malattie genetiche e, addirittura, a medio termine sradicarle dalla nostra società. Le enormi potenzialità terapeutiche di questa tecnica hanno addirittura attirato l’attenzione dell’Accademia Reale Svedese delle Scienze che, proprio mentre si stanno scrivendo queste righe, ha attribuito alle sue inventrici, Jennifer Doudna ed Emmanuelle Charpentier, il Premio Nobel per la Chimica 2020, definendo CRISPR/Cas9 come “un rivoluzionario metodo di editing genetico che contribuisce allo sviluppo di nuove terapie contro il cancro e può realizzare il sogno di curare malattie ereditarie” (The Royal Swedish Academy of Sciences 2020a). Al fianco di queste prospettive, che dal 2017 ad oggi si sono fatte sempre più evidenti, si annidano però rischi e pericoli derivanti dall’uso delle tecniche d’ingegneria genetica che il diritto deve tenere in adeguata considerazione al momento della loro regolamentazione. Nei primi mesi di lavoro, dedicati proprio alla ricostruzione delle fonti giuridiche applicabili, ci ha subito colpito che nonostante le tecniche in parola costituiscano, ancora oggi, un’assoluta novità in continuo cambiamento, le norme giuridiche, sia sovranazionali che nazionali, siano relativamente risalenti nel tempo: la legge 40 che, in Italia, si propone di regolare la procreazione medicalmente assistita e alla lett. b) del co. 3 del suo art. 13 si occupa delle manipolazioni genetiche è del 2004, mentre la norma più rilevante sul punto a livello internazionale, l’art. 13 della Convenzione di Oviedo, è addirittura datata aprile 1997. Insomma, in questo campo il diritto anziché presentarsi in fisiologico ritardo, ha enucleato delle regolamentazioni in sospetto anticipo. Questa constatazione, combinata con gli esiti della ricostruzione del dibattito dottrinale, dove anche autorevolissimi autori combinano continuamente argomentazioni etiche ed argomentazioni giuridiche, spesso senza neppure differenziarle, ci ha condotto ad appurare come prima di affrontare il tema della regolamentazione specifica del genome editing fosse necessario riflettere su come il diritto s’interfacci innanzi al bios come oggetto normativo e, soprattutto, in quale relazione si ponga con la bioetica nell’espletare siffatta funzione. Pertanto, abbiamo deciso di dedicare la Parte Prima dell’opera proprio ad un’indagine sulla relazione tra la bioetica ed il biodiritto, che costituiscono la proiezione applicativa di etica e diritto al bios, finalizzata a dotare di un adeguato fondamento epistemologico l’intuizione della deriva di de-differenziazione tra essi. Per raggiungere tale obiettivo abbiamo ritenuto necessario partire, nel Capitolo I, da una breve genealogia della bioetica in cui ci siamo interrogati sulla nascita di questa disciplina e sulle sue successive svolte metodologiche. Il Capitolo II, invece, è stato dedicato alle origini di quello specifico ambito della comunicazione giuridica, comunemente identificato ormai come biodiritto, mettendo in evidenza i contributi interni che la scienza giuridica ha fornito per lo sviluppo dello stesso e riflettendo, in particolare, sul ruolo che ha giocato in tal senso l’istituzione giuridica dei diritti umani. Al contrario, il Capitolo III parte dai contributi esterni alla nascita del biodiritto e specificatamente quelli forniti dalla bioetica per proseguire, poi, con una riflessione sul rapporto tra questi. Mediante una ricostruzione delle posizioni dominanti in dottrina e soprattutto attraverso uno sguardo fisso alla prassi, si è posto in evidenza come, ad oggi, via sia un problema di de-differenziazione tra bioetica e biodiritto che ha portato quest’ultimo a trasformarsi in una scienza ancillare alla prima al punto da essere definito come “diritto della bioetica”. Lungi dal fermarci su posizioni unicamente critiche, abbiamo dotato l’ultima parte del Capitolo di una pars construens in cui abbiamo evidenziato i vantaggi di una relazione funzionalmente differenziata tra bioetica e biodiritto, senza però trascurare anche i problemi ad essa sottesi. Con il chiaro intento di testare i nostri approdi teorici nell’esperienza empirica e, allo stesso tempo, per assolvere all’intento originario della nostra opera, abbiamo deciso di dedicare la Parte II interamente alle implicazioni etiche, sociologiche e giuridiche derivanti dalle tecniche di manipolazione genetica germinale. Per farlo si è reso necessario, anzi tutto, dedicare il Capitolo IV a comprendere, tecnicamente, cosa sia una modificazione genetica germinale e quali siano le posizioni rinvenibili all’interno della comunità scientifica. Il Capitolo V, invece, è stato dedicato ad affrontare i problemi, i rischi, le promesse e le speranze che si annidano intorno alla nostra tecnica: dal timore per una deriva eugenetica alla compatibilità delle modificazioni con l’autocomprensione e la dignità del genere umano, passando per le preoccupazioni delle comunità delle persone diversamente abili e dei genitori, che rischiano di restare schiacciati dalle pressioni sociali, giungendo a prendere in seria considerazione però anche le possibilità di sradicare odiose malattie genetiche una volta per tutte, liberando l’umanità di alcune atroci sofferenze. Con un quadro chiaro dei diversi valori che mette in gioco ed in potenziale conflitto tra loro la tecnica germinale, abbiamo finalmente affrontato il problema della regolamentazione delle nostre tecniche. Abbiamo cercato di farlo non con l’animo di produrre una mera attività compilativa sulle regolamentazioni esistenti e neanche con il solo intento di mostrare lacune e paradossi che in esse si annidano, ma con la finalità più ambiziosa di verificare se le nostre conclusioni teoriche della Parte Prima fossero fondate: se effettivamente il diritto si propone come un mero trasformatore permanente di principi bioetici in precetti coercitivi e se l’approccio regolativo vigente sia adeguato per cogliere i benefici che una tecnica premiata con il Nobel per la Chimica può dare alla società, senza rinunciare a tutelare i diritti fondamentali delle persone.

Dissertação de Mestrado em Direito apresentada à Faculdade de Direito
O mundo tem vindo a assistir grandes desenvolvimentos no domínio da genética e medicina reprodutiva, passando-se a falar da “Revolução GNR (Genética, Nanotecnologia e Robótica)”, capaz de promover a saúde e qualidade de vida humana, como nunca antes. O avanço que maior destaque tem tido na comunidade científica e que será aqui objeto de estudo, insere-se no contexto da Engenharia genética, com o surgimento da tecnologia CRISPR/Cas com potencialidade de corrigir, substituir e modificar o genoma humano, de forma rápida e precisa, visando o aprimoramento genético e/ou a prevenção e tratamento de doenças/malformações genéticas. Contudo, com ela surgem também riscos que colocam em dúvida a sua utilização no contexto da prática clínica, reclamando o debate público, a sua regulamentação e o estabelecimento de critérios a serem seguidos caso o seu uso venha a ser admitido. Não obstante, várias são as normas internacionais, supranacionais e nacionais, com princípios norteadores da investigação científica e prática clínica, no contexto da genética e da biomedicina, que iremos evidenciar. Com base nessa análise, passaremos para a consideração dos dilemas ético-jurídicos que surgem à volta da terapia génica germinal e que se prendem com direitos fundamentais do ser humano. E, sendo esta uma realidade cada vez mais próxima, importa a reflexão acerca da responsabilidade civil dos médicos, por danos que possam surgir no âmbito da terapia génica germinal, analisando os seus pressupostos, focando-nos no domínio privado, e na consequente propositura das wrong actions e surgimento das novas ações de wrongful genetic makeup. Neste caminho, refletimos ainda acerca do eventual surgimento de novos direitos e danos daí decorrentes, fazendo, por fim, breve reflexão sobre os prazos de prescrição, tendo em conta a incerteza e tardia manifestação desses danos. Concluímos defendendo a admissibilidade da terapia génica germinal, ainda que após debate público, reflexão sobre a responsabilidade civil dos profissionais de saúde pelas lesões que daí possam surgir, e regulamentação e fixação de critérios que garantam a segurança das técnicas.
The world has been witnessing great developments in the field of genetics and reproductive medicine, arising the “GNR (Genetics, Nanotechnology and Robotics) Revolution”, capable of promoting human health and quality of life like never before. The most prominent advance in the scientific community which will be the object of study here is part of the context of genetic engineering, which is the emergence of the CRISPR/Cas technology with the potential to correct, replace and modify the human genome, in a more precise and faster way, aiming at genetic enhancement and/or the prevention and treatment of genetic diseases/malformations. However, with it arises risks that cast doubt on its use in the context of clinical practice, demanding public debate, its regulation, and the establishment of criteria to be followed if its use is admitted. Nevertheless, there are several international, supranational and national norms, with guiding principles for scientific research and clinical practice, in the context of genetics and biomedicine, which will be highlighted.Based on this analysis, we will move on to the consideration of the ethical-juridical dilemmas that arise around germinal gene therapy and that relate to fundamental human rights. And, as this reality is ever closer, it is important to reflect on the civil liability of physicians, for damages that may arise in the context of germinal gene therapy, analyzing its assumptions, focusing on the private domain, and the consequent proposition of wrong actions and the emergence of new wrongful genetic makeup actions. On this path, we also reflect on the possible emergence of new rights and damages arising from these techniques, finally making a brief reflection on the limitation periods, considering the uncertainty and late manifestation of these damages. We conclude defending the admissibility of germinal gene therapy, only after a public debate, reflection on civil liability of health professionals for the damages that may arise from it, and fixation of criteria that guarantee the safety of the techniques.

Gene targeting (GT) is a much needed technology as a tool for plant research and for the precise engineering of crop species. Recent advances in this field have shown that the presence of a DNA double-strand break (DSB) in a genomic locus is critical for the integration of an exogenous DNA molecule introduced into this locus. This integration can occur via either non-homologous end joining (NHEJ) into the break or homologous recombination (HR) between the broken genomic DNA and the introduced vector. A bottleneck for DNA integration via HR is the machinery responsible for homology search and strand invasion. Important proteins in this pathway are Rad51, Rad52 and Rad54. We proposed to combine our respective expertise: on the US side, in the design of zincfinger nucleases (ZFNs) for the induction of DNA DSBs at any desired genomic locus and in the integration of DNA molecules via NHEJ; and on the Israeli side in the HR events, downstream of the DSB, that lead to homology search and strand invasion. We sought to test three major pathways of targeted DNA integration: (i) integration by NHEJ into DSBs induced at desired sites by specially designed ZFNs; (ii) integration into DSBs induced at desired sites combined with the use of Rad51, Rad52 and Rad54 proteins to maximize the chances for efficient and precise HR-mediated vector insertion; (iii) stimulation of HR by Rad51, Rad52 and Rad54 in the absence of DSB induction. We also proposed to study the formation of dsT-DNA molecules during the transformation of plant cells. dsT-DNA molecules are an important substrate for HR and NHEJ-mediatedGT, yet the mode of their formation from single stranded T-DNA molecules is still obscure. In addition we sought to develop a system for assembly of multi-transgene binary vectors by using ZFNs. The latter may facilitate the production of binary vectors that may be ready for genome editing in transgenic plants. ZFNs were proposed for the induction of DSBs in genomic targets, namely, the FtsH2 gene whose loss of function can easily be identified in somatic tissues as white sectors, and the Cruciferin locus whose targeting by a GFP or RFP reporter vectors can give rise to fluorescent seeds. ZFNs were also proposed for the induction of DSBs in artificial targets and for assembly of multi-gene vectors. We finally sought to address two important cell types in terms of relevance to plant transformation, namely GT of germinal (egg) cells by floral dipping, and GT in somatic cells by root and leave transformation. To be successful, we made use of novel optimized expression cassettes that enable coexpression of all of the genes of interest (ZFNs and Rad genes) in the right tissues (egg or root cells) at the right time, namely when the GT vector is delivered into the cells. Methods were proposed for investigating the complementation of T-strands to dsDNA molecules in living plant cells. During the course of this research, we (i) designed, assembled and tested, in vitro, a pair of new ZFNs capable of targeting the Cruciferin gene, (ii) produced transgenic plants which expresses for ZFN monomers for targeting of the FtsH2 gene. Expression of these enzymes is controlled by constitutive or heat shock induced promoters, (iii) produced a large population of transgenic Arabidopsis lines in which mutated mGUS gene was incorporated into different genomic locations, (iv) designed a system for egg-cell-specific expression of ZFNs and RAD genes and initiate GT experiments, (v) demonstrated that we can achieve NHEJ-mediated gene replacement in plant cells (vi) developed a system for ZFN and homing endonuclease-mediated assembly of multigene plant transformation vectors and (vii) explored the mechanism of dsTDNA formation in plant cells. This work has substantially advanced our understanding of the mechanisms of DNA integration into plants and furthered the development of important new tools for GT in plants.