Littérature scientifique sur le sujet « Editing genetico 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.

Arguably the human body has been one of the most sophisticated systems we encounter but until now we are still far from understanding its complexity. We have been trying to replicate human intelligence by way of artificial intelligence but with limited success. We have discovered the molecular structure in terms of genetics, performed gene editing to change an organism’s DNA and much more, but their translatability into the field of oncology has remained limited. Conventional machine learning methods achieved some degree of success in solving problems that we do not have an explicit algorithm. However, they are basically shallow learning methods, not rich enough to discover and extract intricate features that represent patterns in the real environment. Deep learning has exceeded human performance in pattern recognition as well as strategic games and are powerful for dealing with many complex problems. High-throughput sequencing and microarray techniques have generated vast amounts of data and allowed the comprehensive study of gene expression in tumor cells. The application of deep learning with molecular data enables applications in oncology with information not available from clinical diagnosis. This paper provides fundamental concepts of deep learning, an essential knowledge of cancer genetics, and a review of applications of deep learning to cancer oncology. Importantly, it provides an insightful knowledge of deep learning and an extensive discussion on its challenges. The ultimate purpose is to germinate ideas and facilitate collaborations between cancer biologists and deep learning researchers to address challenging oncological problems using advanced deep learning technologies.

Abstract Most B cell non-Hodgkin’s lymphomas (B-NHL) derive from germinal center (GC) B cells and their pathogenesis is associated with the accumulation of distinct genetic lesions, including chromosomal translocations and a more recently identified mechanism of genomic instability, termed aberrant somatic hypermutation. These alterations are thought to be due to mistakes occurring during two GC-associated immunoglobulin (Ig) genes remodeling processes: class switch recombination (CSR) and somatic hypermutation (SHM). However, this model has never been formally proven. To conclusively investigate the role of CSR and SHM in the pathogenesis of B-NHL, we examined whether lymphoma development in mice requires the function of activation induced cytidine deaminase (AID), a DNA editing enzyme expressed specifically in GC and activated B cells and essential for both processes. Three transgenic mouse models were generated by crossing lymphoma-prone mice (λMYC, λMYC/IμHABCL6 and IμHABCL6) with mice (AID−/−) that are unable to undergo both SHM and CSR. The λMYC mice develop a diffusely infiltrating monoclonal proliferation of pre-GC origin, with unmutated IgV genes and lack of BCL6 expression, and therefore presumably independent from AID-associated DNA remodeling events. Conversely, lymphomas in λMYC/IμHABCL6 and IμHABCL6 mice recapitulate GC/post GC-derived malignancies, in that the former display somatically mutated IgV genes and upregulation of post-GC markers (CD138) in most of the cases, while the latter develop a splenic lymphoproliferative syndrome that culminates, past 12 months of age, in clonal B cell lymphomas with DLBCL morphology and somatically mutated IgV genes (~70% of the animals) (Cattoretti et al., Cancer Cell 7:445–455, 2005). Mice were monitored for tumor incidence and survival, and a combination of histologic, immunophenotypic and gene expression profiling analysis was used for tumor characterization. As expected, no significant differences in event-free survival and lymphoma type were observed between AID-proficient and AID-deficient λMYC mice, in agreement with their pre-GC derivation. Conversely, a phenotypic shift of the tumor was observed in λMYC/IμHABCL6 mice when bred into an AID−/− background, with >80% of the cases (N=21/26) reverting to a pre-GC phenotype (loss of GC/post GC markers) undistinguishable from that of the λMYC and λMYC/AID−/− mice. Gene expression profile analysis on representative cases (N=10 λMYC/IμHABCL6 and 5 each for λMYC, λMYC/AIDKO, λMYC/IμHABCL6/AIDKO) confirmed significant phenotypic similarities between pre-GC derived λMYC lymphomas and the λMYC/IμHABCL6/AID −/− lymphomas, which co-segregated in a separate cluster from λMYC/IμHABCL6 tumors. Analogously, a significant reduction in DLBCL frequency was observed in the IμHABCL6/AIDKO cohort as compared to IμHABCL6 mice (N= 4/19, 21% vs 8/14, 57%; p=0.03). Taken together, these results indicate that GC-derived lymphomas cannot develop in the absence of AID, thereby providing direct support to the notion that AID-mediated mistakes in antigen receptor gene modification events (CSR and SHM) represent major contributors to B-NHL pathogenesis.

Axon injury is a hallmark of many neurodegenerative diseases, often resulting in neuronal cell death and functional impairment. Dual leucine zipper kinase (DLK) has emerged as a key mediator of this process. However, while DLK inhibition is robustly protective in a wide range of neurodegenerative disease models, it also inhibits axonal regeneration. Indeed, there are no genetic perturbations that are known to both improve long-term survival and promote regeneration. To identify such a neuroprotective target, we conducted a set of complementary high-throughput screens using a protein kinase inhibitor library in human stem cell-derived retinal ganglion cells (hRGCs). Overlapping compounds that promoted both neuroprotection and neurite outgrowth were bioinformatically deconvoluted to identify specific kinases that regulated neuronal death and axon regeneration. This work identified the role of germinal cell kinase four (GCK-IV) kinases in cell death and additionally revealed their unexpected activity in suppressing axon regeneration. Using an adeno-associated virus (AAV) approach, coupled with genome editing, we validated that GCK-IV kinase knockout improves neuronal survival, comparable to that of DLK knockout, while simultaneously promoting axon regeneration. Finally, we also found that GCK-IV kinase inhibition also prevented the attrition of RGCs in developing retinal organoid cultures without compromising axon outgrowth, addressing a major issue in the field of stem cell-derived retinas. Together, these results demonstrate a role for the GCK-IV kinases in dissociating the cell death and axonal outgrowth in neurons and their druggability provides for therapeutic options for neurodegenerative diseases.

Epstein–Barr virus (EBV), defined as a group I carcinogen by the World Health Organization (WHO), is present in the tumour cells of patients with different forms of B-cell lymphoma, including Burkitt lymphoma, Hodgkin lymphoma, post-transplant lymphoproliferative disorders, and, most recently, diffuse large B-cell lymphoma (DLBCL). Understanding how EBV contributes to the development of these different types of B-cell lymphoma has not only provided fundamental insights into the underlying mechanisms of viral oncogenesis, but has also highlighted potential new therapeutic opportunities. In this review, we describe the effects of EBV infection in normal B-cells and we address the germinal centre model of infection and how this can lead to lymphoma in some instances. We then explore the recent reclassification of EBV+ DLBCL as an established entity in the WHO fifth edition and ICC 2022 classifications, emphasising the unique nature of this entity. To that end, we also explore the unique genetic background of this entity and briefly discuss the potential role of the tumour microenvironment in lymphomagenesis and disease progression. Despite the recent progress in elucidating the mechanisms of this malignancy, much work remains to be done to improve patient stratification, treatment strategies, and outcomes.

Mouse models represent invaluable tools for the systematic evaluation of cancer drivers, yet models that address the impact of putative genetic drivers of chronic lymphocytic leukemia (CLL) on B cell development and function are largely lacking. To study recurrent loss-of-function (LOF) mutations observed in human CLL, we established a transplant model that can rapidly evaluate genetic lesions. First, we crossed mice carrying B-cell restricted Cre expression (Cd19-cre) with mice carrying conditional Cas9-GFP, to generate a strain expressing B cell-restricted Cas9 (Cd19-Cas9). Next, we optimized methods for in vitro engineering of early stem and progenitor cells (Lin- Sca-1+ c-kit+ [LSK]) from Cd19-Cas9 mice using lentivirus expressing sgRNAs (mCherry+)targeting Atm, Trp53, Chd2, Birc3, Mga, or Samhd1. We chose LSKs because of their high transducibility and long-term repopulating potential. Last, we transplanted the single sgRNA-expressing LSKs into sub-lethally irradiated CD45.1 recipient mice, and then confirmed presence of ~45-85% gene-edited sequences (>70% carrying frameshift mutations) in edited B cells (GFP+mCherry+) at 2 months post-transplant, by PCR-based targeted deep sequencing and CRISPResso software analysis. We also verified presence of gene alterations (and putative off-target lesions) at the single cell DNA level (targeted sequencing by Tapestri, Mission Bio). We first asked whether presence of the 6 LOFs could impact B cell developmental trajectories in marrow, spleen and peritoneum at 4 months post-transplant, a time point by which B cells are considered to achieve optimal host reconstitution (n=5/group, including a non-targeting control group). No marked changes were observed in mice with Atmindel, Trp53indel, Chd2indel, Birc3indel or Samhd1indel, as analyzed by flow cytometry. Of interest, however, Mgaindel mice were detected to have increased germinal center (B220+CD95+CD38-) and marginal zone (B220+CD21highCD23-) splenic B cells, and also showed increased B1a (CD5+ B220low CD23- CD43+) and decreased B1b (CD5- B220low CD23- CD43+) cells in the peritoneum (p<0.05, ANOVA). These results indicate that the likely negative regulatory role that Mga exerts on MYC networks may directly impact germinal center formation and cell fate determination in B cells. The overall abundance of edited B cells in spleen and blood of each group was higher (overall median: 17.0%; 90%CI 6.7-58.8%) than the non-targeting control (8.4%; 90%CI 1.6-14.2%) at 4 months post-transplant (n=8/group, p<0.05, ANOVA), and abundance of edited cells increased in peripheral bleeds at 4 vs. 2 months (n=8/group, p<0.05, Wilcoxon signed rank test). This suggests that presence of individual alterations can alter pro-survival pathways in mature B cells, through mechanisms that may, at least partly, be shared across LOFs. To address this question, we analyzed the transcriptional profiles of edited B cell splenocytes (n=3/group), and compared them to their non-edited counterparts (GFP+mCherry- splenocytes from the same animal), identifying a total of ~3900 differentially expressed genes among the 6 groups (p<0.05, paired Student's t test). Notably, changes in gene expression were highly concordant across 5 of the 6 groups (Spearman r >0.37 for each of the 10 pairs of 5 groups), with the exception of Mgaindel, consistent with its unique phenotype, observed in developmental studies. Gene ontology analyses using Enrichr confirmed commonalities in pathway dysregulations across the 5 similar groups of mice (p<0.05), such as modulation of Notch signaling in Chd2indel, Samhd1indel, and Birc3indel, serine/glycine metabolism in Atmindel, Trp53indel, and Chd2indel, and oxidative phosphorylation in Atmindel and Samhd1indel. Unique to Mgaindel, we saw enrichment of the GOs for transcriptional mis-regulation in cancer and cellular senescence, both relevant for tumorigenesis and B cell development. In conclusion, we demonstrate that common LOFs typical of patients with CLL lead to increased cellular fitness in B-cell restricted mouse models, while dysregulating pro-survival pathways relevant to B cell development, CLL pathogenesis and more broadly to tumorigenesis. We are currently exploring phenotypic similarities and differences through tailored functional assays, while addressing the relative contribution of each alteration to CLL development in multiplexed edited mouse lines. Disclosures Wang: Mission Bio Inc.: Employment. Jacob:Mission Bio Inc.: Employment. Flynn:Mission Bio Inc.: Employment. Ruff:Mission Bio Inc.: Employment. Jones:Mission Bio Inc.: Employment. Neuberg:Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding.

Abstract Background: The current standard of care in diffuse large B-cell lymphoma (DLBCL) consists of chemotherapy and therapeutic monoclonal antibodies that have significantly improved patient outcomes over the past 15 years. However, a large proportion of patients suffer from refractory or relapsed disease. Therefore, the development of new therapeutic strategies for this subgroup of patients, who are threatened by a high chance of disease-related death, represents an important unmet clinical need. Methods: We enrolled into our study 347 de novo DLBCL patients uniformly treated with R-CHOP from the BC Cancer population-based cohort between September 2000 and January 2012. RNAseq and high-resolution copy number analysis were performed and correlated with clinical outcome data and tumor microenvironment composition. We also performed functional studies to investigate PRAME-mediated memory T-cell responses and gene expression changes. Results: We discovered novel, highly focal deletions of 22q11.22, including the PRAME gene in 13% (44/338) of the cases. The deletions cluster in a narrow chromosomal region that includes a very small number of genes (VpreB1, ZNF280A/B, PRAME, GGTLC2, miR-650). Of clinical importance, 22q11.22 deletions were found significantly more frequently in germinal centre B-cell-like (GCB) type DLBCL (17% (31/180) vs. activated B-cell-like (ABC) type: 8% (8/98), P < 0.01), and were also significantly associated with worse outcome, which was specifically observed in GCB-DLBCL (5-year disease specific survival, non-PRAME-deleted: 84.5% vs. PRAME-deleted: 67.2%, P = 0.026). Homozygous deletions were more strongly associated with poor outcome than heterozygous deletions. Interestingly, 90% of PRAME-deleted cases were Ig-lambda restricted (P < 0.001). PRAME is a prominent member of the cancer testis antigen (CTA) family of proteins that are expressed in various types of cancers, but not in normal tissues, including normal mature B-cells, apart from male germinal cells. Due to the cancer-specific expression of CTAs, these molecules are considered promising targets for cancer immunotherapy using cytotoxic T-cells and tumor vaccination approaches. To determine the association with tumor microenvironment composition, we analyzed CD4/CD8 flow cytometry data from DLBCL patient samples. The numbers of CD4 and CD8-positive T cells were significantly lower in PRAME-deleted cases compared to wild type (CD4: P < 0.001, CD8: P = 0.013). Notably, RNAseq analysis revealed that the HLA-A*0201 genotype was seen significantly more often in PRAME deleted cases (PRAME wt: 2.5% vs. PRAME deleted: 10.8%, P = 0.005). In order to functionally characterize its interaction with the immune microenvironment, we utilized enzyme-linked immunoSpot (ELISPOT) assays to investigate memory T-cell reactions of patient-derived T cells to PRAME antigens using patient-derived peripheral blood mononuclear cells (PBMC) and measured IFN-g production (7 control healthy donors, 4 PRAME-deleted and 4-wild type patients). While T cells from PRAME-replete patients had no reaction to PRAME antigens, PRAME-deleted patient-derived T-cells had significant reactions to 4 independent PRAME peptides. These data suggest that PRAME-deleted tumor cells can escape from cytotoxic T-cell attack to gain growth advantage. Next, we performed PRAME knock-out (KO) experiments using CRISPR/Cas9 genome editing to clarify the cell autonomous effects of PRAME deletions. Using 2 different cell lines (Karpas422 and SUDHL-4), we found TNFSF10 (TRAIL) expression was significantly down-regulated in homozygous PRAME-KO cell lines compared to wild type. The soluble form of TRAIL (sTRAIL) was also reduced, as measured with enzyme-linked immunosorbent assays. These results suggest that PRAME downregulated cells may contribute to cell survival via TRAIL and sTRAIL reduction. Conclusion: We identified recurrent PRAME deletions and characterized their clinical and functional role in DLBCL. Our findings contribute to the understanding of cell-autonomous and extrinsic roles of PRAME deletions in lymphomagenesis and may lead to the discovery of new therapeutic avenues to simultaneously treat the tumor and the host. Disclosures Gascoyne: NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:Janssen: Research Funding; Roche: Research Funding; NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Celgene: Consultancy, Honoraria. Steidl:Tioma: Research Funding; Seattle Genetics: Consultancy; Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Juno Therapeutics: Consultancy; Nanostring: Patents & Royalties: patent holding.

Histones play an important role in DNA’s compaction and organisation into the cellular nucleus. Depending on which histone modification occurs, chromatin can take a conformation of heterochromatin or euchromatin, which are associated with gene repression or expression, respectively. Histone H3 lysine 9 (H3K9) trimethylation (H3K9me3) is associated with gene silencing. At least 3 methyltransferases are able to change the methylation status of H3K9: SUV39H1, SUV39H2, and SETDB1. In several mammalian species, modulation of H3K9 methylation status has been demonstrated to be necessary to achieve a successful pre-implantation embryonic development after IVF or somatic cell NT. The aim of this work was to study the role of H3K9me3 in IVF pre-implantation bovine embryos. For this purpose, immunostaining of H3K9me3 at different pre-implantation stages of development was performed. Further, the relative abundances of the methyltransferases SUV39H1 and SUV39H2 were measured by real-time PCR using luciferase transcript as an exogenous gene for normalization. Finally, to evaluate H3K9me3 involvement during pre-implantation embryonic development, we generated SUV39H1 or SUV39H2 knockout embryos by the CRISPR/Cas9 system. We designed guide RNA targeting SUV39H1 or SUV39H2 and co-injected the presumptive zygote’s cytoplasm 18h post-fertilization with Cas9 protein. At Day 8 post-fertilization, the number of blastocysts was assessed and embryos were immunostained to evaluate H3K9me3. Results were analysed using Student’s t-test or ANOVA with the post-hoc Tukey test depending on data set (P ≤ 0.05) and reported as means±standard errors of the mean. Oocytes at germinal vesicle stage and metaphase II as well as embryos at different stages of pre-implantation development (2, 4, and 8 cells, morula, and blastocyst; n=6) were immunoreactive for H3K9me3. Expression of SUV39H1 and SUV39H2 mRNA decreased significantly as embryonic development progressed, reaching undetectable levels at stages where genome activation had already occurred (morula and blastocyst; P&lt;0.0001, n=3). When zygotes were co-injected with the guide RNA targeting SUV39H1/Cas9, embryonic production showed a significant increase compared with the control [42.26%±5.03 (28/65) v. 23.86%±3.99 (21/88), respectively; P=0.034, n=4], and H3K9me3 immunostaining was reduced in treated embryos. Editing efficiency was estimated at 66%. In contrast, no statistical differences were found in embryonic production or H3K9me3 immunostaining in embryos co-injected with the guide RNA targeting SUV39H2/Cas9 (P=0.57, n=3). In conclusion, we were able to characterise H3K9me3 and determine transcript levels of methyltransferases SUV39H1 and SUV39H2 in oocytes and different stages of pre-implantation embryonic development. We also demonstrated that SUV39H1 deletion led to an increased embryonic production, suggesting that H3K9me3 removal would allow a greater relaxation of the heterochromatin and consequently a successful activation of embryonic genes. This highlights the essential role of H3K9me3 during bovine pre-implantation embryonic development.

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.

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.