Dissertations / Theses on the topic 'CRISPR, Gene editing, Parkinson, Gene therapy'

Parkinson’s disease (PD) is one of the most common long-term degenerative disorders that affect the nervous system. Clinical symptoms are bradykinesia, resting tremor and postural imbalance due to the loss of dopaminergic neurons in the substantia nigra pars compacta. Heterozygous mutations in the Leucine Rich Repeat Kinase 2 gene (LRRK2) have been identified both in familial and sporadic cases of PD. The most common variant is the p.Gly2019Ser substitution (c.6055G>A). To date there is no effective treatment available. The genome editing tool CRISPR/Cas9 has recently transformed the field of biotechnology and biomedical discovery, posing the basis for the development of innovative treatments. Using CRISPR/Cas9 technology and Homology Directed Repair, our project aims to validate gene editing as an alternative therapeutic approach for PD through the genetic correction of the pathogenic p.Gly2019Ser LRRK2 mutation restoring the wild-type sequence both in human and mouse models. Specifically, we tested various strategies, based on the CRISPR/Cas9-based genome editing technique, for the correction of LRRK2 p.Gly2019Ser (c.6055G>A) variant in primary mouse and human fibroblasts with promising results. If the correction experiments in in vitro models will confirm the good efficiency of the approach, these experiments will represent a fundamental step for the subsequent evaluation of the potential of gene therapy for the treatment of PD as well as other brain disorders for which no therapy is currently available.

Neuroblastoma is a tumour originating from the sympathetic nervous system, and represents the most common extracranial solid cancer in childhood. Despite the malignancy is extremely heterogeneous, about 25% of all cases is characterized by MYCN-gene amplification, aggressive tumour and poor survival. The network of genes that are deregulated in this group of patients represents a focal point for targeted-therapy discovery. Along this research line, the first objective of the present project was to investigate the prognostic significance of a single nucleotide polymorphism (SNP) located in the promoter of ODC1, a neuroblastoma prognostic marker involved in polyamine biosynthesis. The SNP genotype was first associated with survival of a large cohort of patients with aggressive neuroblastoma. Then, CRISPR-editing revealed that the SNP genotype affects ODC1 expression and proliferation of neuroblastoma cells. At last, the SNP was found to influence cell sensibility to DFMO, an ODC1 inhibitor that is currently under trial for treatment of aggressive neuroblastoma. The second objective was to investigate the role in neuroblastoma development and progression of RUNX1T1, a poorly studied transcription repressor involved in distinct development events and cancers. Survival analysis of a cohort of neuroblastoma patients revealed that RUNX1T1 is a potential oncosuppressor. In apparent contrast, RUNX1T1 knockout by CRISPR-editing demonstrated that the gene promotes aggressiveness of neuroblastoma cells. Transcriptome analysis of the mutant cells then evidenced deregulation of a significant number of genes and pathways that are prognostic markers in neuroblastoma, therefore depicting a multifunctional regulation network that could be exploited for new therapies. The third and last objective was to test a novel therapeutic approach based on MYCN-amplification targeting via CRISPR-cleavage. In vitro experiments demonstrated that the system efficiently and specifically impairs the survival of aggressive neuroblastoma cells, thus providing a proof of principle for the development of an innovative therapy.

The human genome encodes information that instructs human development, physiology, medicine, and evolution. Massive amount of genomic data has generated an ever-growing pool of hypothesis. Genome editing, broadly defined as targeted changes to the genome, posits to deliver the promise of genomic revolution to transform basic science and personalized medicine. This thesis aims to contribute to this scientific endeavor with a particular focus on the development of effective human genome engineering tools.

Several tools were developed to help researchers facilitate clinical translation of the use of engineered nucleases towards their disease gene of interest. Two major issues addressed were the inability to accurately predict nuclease off-target sites by user-friendly \textit{in silico} methods and the lack of a high-throughput, sensitive measurement of gene editing activity at endogenous loci. These objectives were accomplished by the completion of the following specific aims. An online search interface to allow exhaustive searching of a genome for potential nuclease off-target sites was implemented. Previously discovered off-target sites were collated and ranking algorithms developed that preferentially score validated off-target sites higher than other predictions. HEK-293T cells transfected with newly developed TALENs and ZFNs targeting the beta-globin gene were analyzed at the off-target sites predicted by the tool. Many samples of genomic DNA from cells treated with different ZFNs and TALENs were analyzed for off-target effects to generate a greatly expanded training set of bona fide off-target sites. Modifications to the off-target prediction algorithm parameters were evaluated for improvement through Precision-Recall analysis and several other metrics. An analysis pipeline was developed to process SMRT reads to simultaneously measure the rates of different DNA repair mechanisms by directly examining the DNA sequences. K562 cells were transfected with different types of nucleases and donor repair templates in order to optimize conditions for repairing the beta-globin gene. This work will have significant impact on future studies as the methods developed herein allow other laboratories to optimize nuclease-based therapies for single gene disorders.

Gene repair involves the correction of the genetic mutation directly at the defective locus with retention of physiological expression. The biggest challenge of this approach, however, is that gene repair by homologous recombination occurs at levels that are unlikely to be sufficient to confer therapeutic benefit in the majority of cell-autonomous liver disease phenotypes, such as OTC deficiency, the most common urea cycle disorder. To overcome this challenge, gene correction can be complemented by selective expansion strategies designed to expand repaired hepatocytes to frequencies required for therapeutic benefit. In vivo expansion can be achieved, for instance, by conferring a selective advantage to gene-corrected cells. In this study, human-specific genetic inhibitors were designed to exploit a selective expansion strategy based on the modulation of the tyrosine catabolism pathway and were successfully validated in humanised (Fah-/-, Rag2-/-, IL2rg-/-) FRG mice. Another way to increase the frequency of gene repair is to use nucleases to create DNA breaks at the target site to promote homology-directed repair (HDR). Recombinant AAV vectors carrying human-specific reagents for CRISPR/Cas9-mediated genome editing were developed in order to correct a single nucleotide mutation in exon 9 of the OTC gene. Initially, the editing reagents were evaluated in OTC-deficient mice with a transposed engineered “minigene” version of the OTC gene. Editing reagents functionally validated in this model were then evaluated in vivo on the native OTC locus in primary human hepatocytes, including patient-derived hepatocytes, xenografted into FRG mice. Availability of novel synthetic AAV capsids, such as NP59, facilitated high targeting efficiency of human hepatocytes which in turn resulted in up to 29% OTC alleles being corrected by HDR. The studies described in this thesis show for the first time precise gene repair of a disease-causing mutation in primary human hepatocytes in vivo.

Rare diseases, when considered as a whole, affect up to 7% of the population, which would represent 3.5 million individuals in the United Kingdom alone. However, while 'personalised medicine' is now yielding remarkable results using recent sequencing technologies in terms of diagnosing genetic conditions, we have made much less headway in translating this patient information into therapies and effective treatments. Even with recent calls for greater research into personalised treatments for those affected by a rare disease, progress in this area is still severely lacking, in part due to the astronomical cost and time involved in bringing treatments to the clinic. Gene correction using the recently-described genome editing technology CRISPR/Cas9, which allows precise editing of DNA, offers an exciting new avenue of treatment, if not cure, for rare diseases; up to 80% of which have a genetic component. This system allows the researcher to target any locus in the genome for cleavage with a short guide-RNA, as long as it precedes a highly ubiquitous NGG sequence motif. If a repair sequence is then also provided, such as a wild-type copy of the mutated gene, it can be incorporated by homology-directed repair (HDR), leading to gene correction. As both guide-RNA and repair template are easily generated, whilst the machinery for editing and delivery remain the same, this system could usher in the era of 'personalised medicine' and offer hope to those with rare genetic diseases. However, currently it is difficult to test the efficacy of CRISPR/Cas9 for gene correction, especially in vivo. Therefore, in my PhD I have developed a novel fluorescent reporter system which provides a rapid, visual read-out of both non-homologous end joining (NHEJ) and homology-directed repair (HDR) driven by CRISPR/Cas9. This system is built upon a cassette which is stably and heterozygously integrated into a ubiquitously expressed locus in the mouse genome. This cassette contains a strong hybrid promoter driving expression of membrane-tagged tdTomato, followed by a strong stop sequence, and then membrane-tagged EGFP. Unedited, this system drives strong expression of membrane-tdTomato in all cell types in the embryo and adult mouse. However, following the addition of CRISPR/Cas9 components, and upon cleavage, the tdTomato is rapidly excised, resulting via NHEJ either in cells without fluorescence (due to imperfect deletions) or with membrane-EGFP. If a repair template containing nuclear tagged-EGFP is also supplied, the editing machinery may then use the precise HDR pathway, which results in a rapid transition from membrane-tdTomato to nuclear- EGFP. Thereby this system allows the kinetics of editing to be visualised in real time and allows simple scoring of the proportion of cells which have been edited by NHEJ or corrected by HDR. It therefore provides a simple, fast and scalable manner to optimise reagents and protocols for gene correction by CRISPR/Cas9, especially compared to sequencing approaches, and will prove broadly useful to many researchers in the field. Further to this, I have shown that methods which lead to gene correction in our reporter system are also able to partially repair mutations found in the disease-causing gene, Zmynd10; which is implicated in the respiratory disorder primary ciliary dyskinesia (PCD), for which there is no effective treatment. PCD is an autosomal-recessive rare disorder affecting motile cilia (MIM:244400), which results in impaired mucociliary clearance leading to neonatal respiratory distress and recurrent airway infections, often progressing to lung failure. Clinically, PCD is a chronic airway disease, similar to CF, with progressive deterioration of lung function and lower airway bacterial colonization. However, unlike CF which is monogenic, over 40 genes are known to cause PCD. The high genetic heterogeneity of this rare disease makes it well suited to such a genome editing strategy, which can be tailored for the correction of any mutated locus.

Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of genome function. Analogous to the search function in modern word processors, Cas9 can be guided to specific locations within complex genomes by a short RNA search string. Using this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice. Cas9-mediated genetic perturbation is simple and scalable, empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. To facilitate successful and specific Cas9 targeting, we first optimize the guide RNAs (sgRNA) to significantly enhance gene editing efficiency and consistency. We also systematically characterize Cas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target mutagenesis. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that Cas9-mediated cleavage is unaffected by DNA methylation and that the dosage of Cas9 and sgRNA can be titrated to minimize off-target modification. Additionally, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses. We next demonstrate that Cas9 nickase mutants can be used with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs can reduce off-target activity by over 1,500-fold in human cells. In collaboration with researchers at the University of Tokyo, we further identified a PAM-interacting domain of the Cas9 nuclease that dictates Cas9 target recognition specificity. Finally, we present protocols that provide experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks. Taken together, this work enables a variety of genome engineering applications from basic biology to biotechnology and medicine.

β (HBB) gene, resulting in absence (β0) or deficiency (β+) of β globin chain synthesis. This genetic disorder occurs most frequently in people from Mediterranean countries, such as Italy. In particular, the data indicates that about 12.6% of the Sardinian subjects are carriers of β thalassemia and these are among the highest frequencies of thalassemia genes found in a Caucasian population. In Sardinia, the disease is generally determined by a nonsense mutation at codon 39 (E39X) of exon 2 causing the interruption of β globin synthesis. Patients homozygous with E39X mutation have a severe anemia and require frequent transfusions and iron chelation. The only definitive cure today possible for β chain hemoglobinopathies is the hematopoietic stem cells transplantation, but it is limited by availability of HLA matched donors. However, in the last few years new therapeutic approaches for this genetic disease are taking place. The correction of disease-causing mutation through the technique of Genome-Editing in patient-specific stem cells and subsequent autologous transplantation would be the ideal approach for the treatment of monogenic diseases such as β thalassemia. However, due to difficulties in obtaining sufficient homologous recombination percentages for therapeutic purposes, the aim of my PhD project is to reproduce artificially the HPFH mutations identified in non-coding regions of the β globin cluster, using the system CRISPR/Cas9 associated with NHEJ pathway. In this way, we hope to restore at therapeutic levels the expression of HBG genes and consequently the synthesis of a functional HbF in order to ameliorate the phenotype of β thalassemia.

Mutations in the SOD1 gene are the best characterized genetic cause of amyotrophic lateral sclerosis (ALS) and account for ~20% of inherited cases and 1-3% of sporadic cases. The gene-editing tool Cas9 can silence mutant genes that cause disease, but effective delivery of CRISPR-Cas9 to the central nervous system (CNS) remains challenging. Here, I developed strategies using canonical Streptococcus pyogenes Cas9 to silence SOD1. In the first strategy, I demonstrate effectiveness of systemic delivery of guide RNA targeting SOD1 to the CNS in a transgenic mouse model expressing human mutant SOD1 and Cas9. Silencing was observed in both the brain and the spinal cord. In the second strategy, I demonstrate the effectiveness of delivering both guide RNA and Cas9 via two AAVs into the ventricles of the brain of SOD1G93A mice. Silencing was observed in the brain and in motor neurons within the spinal cord. For both strategies, treated mice had prolonged survival when compared to controls. Treated mice also had improvements in grip strength and rotarod function. For ICV treated mice, we detected a benefit of SOD1 silencing using net axonal transport assays, a novel method to detect motor neuron function in mice before onset of motor symptoms. These studies demonstrate that Cas9-mediated genome editing can mediate disease gene silencing in motor neurons and warrants further development for use as a therapeutic intervention for SOD1-linked ALS patients.

Factor VIII is a clotting factor found on the intrinsic side of the coagulation cascade. A mutation in the factor VIII gene causes the disease Hemophilia A, for which there is no cure. The most common treatment is administration of recombinant factor VIII. However, this can cause an immune response that renders the treatment ineffective in certain hemophilia patients. For this reason a new treatment, or cure, needs to be developed. Gene editing is one solution to correcting the factor VIII mutation. CRISPR/Cas9 mediated gene editing introduces a double stranded break in the genomic DNA. Where this break occurs repair mechanisms cause insertions and deletions, or if a template oligonucleotide can be provided point mutations could be introduced or corrected. However, to accomplish this goal for editing factor VIII mutations, a way to deliver the components of CRISPR/Cas9 into somatic cells is needed. In this study, I confirmed that the CRISPR/Cas9 system was able to create a mutation in the factor VIII gene in zebrafish. I also showed that the components of CRISPR/Cas9 could be piggybacked by vivo morpholino into a variety of blood cells. This study also confirmed that the vivo morpholino did not interfere with the gRNA binding to the DNA, or Cas9 protein inducing the double stranded break.

One of the major challenges facing medicine and drug discovery is the large number of genetic diseases caused by inherited mutations leading to a toxic gain-of-function, or loss-of-function of the disease protein. Microbiology offered a new glimpse of hope to address those disorders with the adaptation of the bacterial CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) defense system as a genome editing tool. Cas9 is a unique CRISPR-associated endonuclease protein that can be easily programmed with an RNA [a single-guide RNA (sgRNA)] that is complementary to nearly any DNA locus. Cas9 creates a double-stranded break (DSB) that can be exploited to knock out toxic genes or replenish therapeutic expression levels of essential proteins. In addition to a matching sgRNA sequence, Cas9 requires the presence of a short signature sequence [a protospacer adjacent motif (PAM)] flanking the target locus. Over the past few years, several Cas9-based therapeutic platforms have emerged to correct DNA mutations in a wide range of mammalian cell lines, ex vivo, and in vivo by adapting recombinant adeno-associated virus (rAAV). However, most of the applications of Cas9 in the field have been limited to Streptococcus pyogenes (SpyCas9), which, in its wild-type form, suffers from inaccurate editing at off-target sites. It is also difficult to deliver via an all-in-one (sgRNA+Cas9) rAAV approach due to its large size. In this thesis, I describe other Cas9 nucleases and their development as new AAV-based genome editing platforms for therapeutic editing in vivo in mouse disease models. In the first part of this thesis, I develop the all-in-one AAV strategy to deliver a Neisseria meningitidis Cas9 ortholog (Nme1Cas9) in mice to reduce the level of circulating cholesterol in blood. I also help characterize an enhanced Cas9 from another meningococcus strain (Nme2Cas9) and show that it is effective in performing editing not only in mammalian cell culture, but also in vivo by all-in-one AAV delivery. Additionally, I describe two AAV platforms that enable advanced editing modalities in vivo: 1) segmental DNA deletion by delivering two sgRNAs (along with Nme2Cas9) in one AAV, and 2) precise HDR-based repair by fitting Nme2Cas9, sgRNA and donor DNA within a single AAV capsid. Using these tools, we successfully treat two genetic disorders in mice, underscoring the importance of this powerful duo of AAV and Cas9 in gene therapy to advance novel treatment. Finally, I present preliminary data on how to use these AAV.Nme2Cas9 vectors to treat Alexander Disease, a rare progressive neurological disorder. These findings provide a platform for future application of gene editing in therapeutics.

IntroductionPreimplantation Genetic Diagnosis (PGD) was developed in the 1990s and has been used since to diagnose and discard embryos with genetic conditions or chromosomal abnormalities. CRISPR-Cas9 was discovered in 2012 and has been used in research, but has not become clinical practice on humans yet. CRISPR-Cas9 could potentially be applied to treat and prevent genetic disorders.AimThe aim was to investigate the ethical dilemmas of each method through a set of research questions. The ethics of applying PGD according to Swedish guidelines and applying CRISPR-Cas9 on humans was investigated.MethodologyThis was not a systematic literature review. Instead, articles have been selected based on their explanation of each method and uniqueness or volume of ethical arguments surrounding each method, that is of relevance for the discussed issues.ResultsArguments in favour of PGD addressed among other things the somatic and psychological health of future children and parents along with the economical benefits. Arguments against PGD addressed different dilemmas of discarding an embryo and thereby a future individual. Arguments against CRISPR-Cas9 addressed technical limitations, our limited knowledge of genetics and more. Arguments in favour addressed benefits in clinical medicine and research.ConclusionsPGD according to Swedish guidelines was found to be ethically acceptable, since its restrictive use that have not given room for ethically dubious applications. CRISPR-Cas9 was found not to be safe enough for human applications at this moment due to technical limitations. If these were to be solved, caution and restraint must be urged.

Gene regulation is a complex and tightly controlled process that defines cell function in physiological and abnormal states. Programmable gene repression technologies enable loss-of-function studies for dissecting gene regulation mechanisms and represent an exciting avenue for gene therapy. Established and recently developed methods now exist to modulate gene sequence, epigenetic marks, transcriptional activity, and post-transcriptional processes, providing unprecedented genetic control over cell phenotype. Our objective was to apply and develop targeted repression technologies for regenerative medicine, genomics, and gene therapy applications. We used RNA interference to control cell cycle regulation in myogenic differentiation and enhance the proliferative capacity of tissue engineered cartilage constructs. These studies demonstrate how modulation of a single gene can be used to guide cell differentiation for regenerative medicine strategies. RNA-guided gene regulation with the CRISPR/Cas9 system has rapidly expanded the targeted repression repertoire from silencing single protein-coding genes to modulation of genes, promoters, and other distal regulatory elements. In order to facilitate its adaptation for basic research and translational applications, we demonstrated the high degree of specificity for gene targeting, gene silencing, and chromatin modification possible with Cas9 repressors. The specificity and effectiveness of RNA-guided transcriptional repressors for silencing endogenous genes are promising characteristics for mechanistic studies of gene regulation and cell phenotype. Furthermore, our results support the use of Cas9-based repressors as a platform for novel gene therapy strategies. We developed an in vivo AAV-based gene repression system for silencing endogenous genes in a mouse model. Together, these studies demonstrate the utility of gene repression tools for guiding cell phenotype and the potential of the RNA-guided CRISPR/Cas9 platform for applications such as causal studies of gene regulatory mechanisms and gene therapy.

Dissertation

Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e Tecnologia
Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3, is a neurodegenerative disorder considered to be the most common form of autosomal dominantly-inherited ataxia in the world. It is a rare disorder, although it has a significative prevalence in some regions of Portugal, especially in the archipelago of Azores. MJD symptoms include a loss of motor coordination and other neurological signs, with the cerebellum being the most affected brain region. MJD arises from an abnormal CAG trinucleotide expansion within the exon 10 of the human ATXN3 gene, which encodes for a protein named ataxin-3 (atxn-3), that bears an aberrant polyglutamine (polyQ) tract in the disease context. The exact biological function of atxn-3 is not fully understood, but it has been described that, when expanded atxn-3 undergoes a toxic gain-of-function that deregulates normal cellular pathways leading to neuronal loss. Recently, it has been suggested that abnormal CAG tracts in mRNA transcripts might also be toxic. As with other polyglutamine diseases, greater numbers of CAG repeats are associated with earlier ages of onset and more severe symptoms.Currently, no therapies capable of delaying or treating the disease are available, and MJD remains a fatal disorder. Many of the therapeutic strategies that have been tested act at a post-transcriptional level, being unable to prevent the putative toxicity of CAG expanded mRNA transcripts. Therefore, new strategies that act at a pre-transcriptional level may be advantageous.However, disease studies can only be robust and feasible when the adequate disease model is used. Yet, the available mouse models used to study MJD either fail to properly mimic the actual genetic context of the disease or are of limited use due to slow and mild symptom progression.In the last decade, the field of gene editing has been thriving. The use of the CRISPR/Cas9 system as a tool for gene editing brought the possibility of performing low-cost, flexible and easy genomic manipulation virtually at any loci. Moreover, CRISPR/Cas9 variants, such as the catalytic inactivated Cas9 (dCas9) have shown promising results regarding gene transcription regulation. In the first part of this work, we planned to establish an in vitro strategy to refine the yeast artificial chromosome (YAC) MJD-Q84.2 mouse, which is the MJD model that best recapitulates the symptoms and the genetic context of the human condition. In this work we proposed to use gene editing tools to overexpand the ATXN3 CAG tract in the exon 10, from 84 to 141 CAG; applying this strategy to the model would be expected to generate a robust mouse model that displayed a more severe phenotype and an earlier disease onset, comparing to the YAC MJD-Q84.2 model. However, the in vitro results here presented, using HEK 293T cells, showed that the strategy used was inefficient at overexpanding the CAG repeats.At the same time, a CRISPR/Cas9-based strategy for correcting the pathogenic ATXN3 gene that could be used to generate isogenic MJD patient-derived cell lines was also tested. The study here presented, using HEK 293T cells, showed that it is possible to integrate a DNA fragment containing 14 CAG repeats plus a selection cassette in the exon 10 of the ATXN3 gene. However, this strategy was not completely infallible, and integration seemed to be independent from Cas9 activity. In the second part of this work, we assessed the effects of dCas9-KRAB as a strategy to pre-transcriptionally silence mutant ATXN3 in a MJD mouse model. In vivo results showed that this approach has a therapeutic potential to improve motor performance in a severely-impaired transgenic mouse model of MJD.
A doença de Machado-Joseph (DMJ), também conhecida por ataxia espinocerebelosa tipo 3, é uma doença neurodegenerativa e a forma mais comum de ataxia hereditária dominante no mundo. É uma doença rara, porém tem uma prevalência significativa em algumas regiões de Portugal, nomeadamente no arquipélago dos Açores. Os sintomas de DMJ incluem perda de coordenação motora bem como outros sinais neurológicos, sendo o cerebelo a região do cérebro mais afetada. A DMJ surge de uma expansão anormal de trinucleótidos CAG no exão 10 do gene ATXN3, que codifica para a proteína ataxina-3 (atxn-3). No contexto da doença, a atxn-3 contém uma sequência de glutaminas (poliQ) anormalmente longa. As funções biológicas da proteína atxn-3 não são totalmente conhecidas, contudo tem sido descrito que a atxn-3 expandida adquire uma função tóxica que desregula o normal funcionamento de diversos sistemas celulares, levando à morte neuronal. Recentemente, tem sido sugerido que tratos CAG anormais presentes em transcritos de mRNA podem também ser tóxicos. Tal como em outras doenças de poliglutaminas, um maior número repetições CAG tem sido associado a sintomas mais severos, com um aparecimento mais precoce.Atualmente, não há terapias capazes de tratar ou atrasar o curso da doença, e, portanto, a DMJ continua a ser fatal. Algumas estratégias terapêuticas que têm sido testadas atuam ao nível pós-transcricional e são por isso incapazes de prevenir os putativos efeitos tóxicos dos transcritos de mRNA que contêm uma cadeia de CAGs expandida. Assim sendo, novas estratégias que atuem a um nível pré-transcricional podem vir a ser vantajosas.O estudo de doenças humanas apenas consegue ser robusto quando é usado um modelo de doença adequado. Porém, os modelos de murganho presentemente usados para estudar a DMJ ou não reproduzem de forma fiel o contexto genético da doença ou apresentam sintomas pouco acentuados e uma progressão lenta que limitam a sua utilização.Na última década, a área da edição genética tem tido desenvolvimentos extraordinários. O uso da CRISPR/Cas9 como uma ferramenta para edição genética trouxe a possibilidade de manipular o genoma virtualmente em qualquer locus, de uma forma barata, fácil e flexível. Além do mais, variantes da CRISPR/Cas9, como é o caso da Cas9 cataliticamente inativa (dCas9), têm mostrado resultados promissores no que diz respeito à regulação da transcrição genética.Na primeira parte deste trabalho pretendemos estabelecer uma estratégia in vitro para aperfeiçoar um modelo de DMJ em murganho designado por yeast artificial chromosome (YAC) MJD-Q84.2, que é considerado como o modelo que melhor recapitula os sintomas e o contexto genético humano em condições de doença. Neste trabalho, utilizámos ferramentas de edição genética para sobreexpandir o trato CAG do exão 10 do gene ATXN3, de 84 para 141 CAGs; ao aplicar esta estratégia ao modelo de murganho seria expectável que se criasse um modelo robusto, com um fenótipo mais severo e em que os sintomas se manifestassem mais cedo em comparação com o já existente modelo YAC MJD-Q84.2. Contudo, os resultados in vitro descritos neste trabalho mostraram que a estratégia usada parece ser ineficiente na sobreexpansão das repetições CAG em células HEK 293T.Paralelamente, usando também a CRISPR/Cas9, foi testada uma estratégia para corrigir o gene ATXN3 mutante, que pudesse ser usada para criar linhas celulares isogénicas derivadas de pacientes de DMJ. O estudo aqui apresentado, usando células HEK 293T, mostrou ser possível integrar um fragmento de DNA contendo 14 repetições do trinucleótido CAG e uma cassete de seleção no exão 10 do gene ATXN3. Contudo, esta abordagem não se mostrou totalmente fiável e que foi detetada integração independente da atividade da Cas9.Na segunda parte deste trabalho foram avaliados os efeitos da dCas9-KRAB como estratégia de silenciamento a nível pré-transcricional do gene humano da ATXN3 num modelo animal de DMJ. Os resultados in vivos mostraram que esta abordagem tem potencial terapêutico, melhorando a performance motora num modelo DJM transgénico severamente afetado.
Outro - This work was funded by the ERDF through the Regional Operational Program Center 2020, Competitiveness Factors Operational Program (COMPETE 2020) and National Funds through FCT (Foundation for Science and Technology) - BrainHealth2020 projects (CENTRO-01-0145-FEDER-000008), ViraVector (CENTRO-01-0145-FEDER-022095), CortaCAGs (PTDC/NEU-NMC/0084/2014|POCI-01-0145-FEDER-016719), SpreadSilencing (POCI-01-0145-FEDER-029716, POCI-01-0145-FEDER-032309) as well as SynSpread, ESMI and ModelPolyQ under the EU Joint Program - Neurodegenerative Disease Research (JPND), the last two co-funded by the European Union H2020 program, GA No.643417; by the National Ataxia Foundation (USA), the American Portuguese Biomedical Research Fund (APBRF) and the Richard Chin and Lily Lock Machado-Joseph Disease Research Fund.

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de Farmácia
The discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), an adaptive defence system for bacteria against viral infections, allowed the creation of a technology called CRISPR/Cas9 system that came to revolutionize genome editing technologies. This system has stood out due to its versatility, simplicity and efficiency in genetic manipulation. Research carried out over the past few years has proven the usefulness and potential of this technology for therapeutic applications, particularly in gene therapy. The potential of the CRISPR/Cas9 system has been progressing thanks to the increasingly detailed knowledge of the mechanisms of this system and the genome editing strategies used. However, there are still many challenges that have to be overcome in order for this technology to be considered safe and effective in the treatment of many pathologies. The discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), an adaptive defence system for bacteria against viral infections, allowed the creation of a technology called CRISPR/Cas9 system that came to revolutionize genome editing technologies. This system has stood out due to its versatility, simplicity and efficiency in genetic manipulation. Research carried out over the past few years has proven the usefulness and potential of this technology for therapeutic applications, particularly in gene therapy. The potential of the CRISPR/Cas9 system has been progressing thanks to the increasingly detailed knowledge of the mechanisms of this system and the genome editing strategies used. However, there are still many challenges that have to be overcome in order for this technology to be considered safe and effective in the treatment of many pathologies.
A descoberta de Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), um sistema de defesa adaptativo de bactérias contra infeções virais, permitiu a criação de uma tecnologia denominada sistema CRISPR/Cas9 que veio revolucionar as tecnologias de edição do genoma. Este sistema tem-se destacado devido à sua versatilidade, simplicidade e eficiência na manipulação genética. As investigações realizadas ao longo dos últimos anos têm vindo a comprovar a utilidade e o potencial desta tecnologia para aplicações terapêuticas, particularmente na terapia génica. O potencial do sistema CRISPR/Cas9 tem vindo a progredir graças ao conhecimento cada vez mais pormenorizado dos mecanismos deste sistema e das estratégias de edição do genoma utilizadas. Existem, no entanto, ainda muitos desafios que têm de ser superados para que esta tecnologia possa ser considerada segura e eficaz no tratamento de muitas patologias. A descoberta de Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), um sistema de defesa adaptativo de bactérias contra infeções virais, permitiu a criação de uma tecnologia denominada sistema CRISPR/Cas9 que veio revolucionar as tecnologias de edição do genoma. Este sistema tem-se destacado devido à sua versatilidade, simplicidade e eficiência na manipulação genética. As investigações realizadas ao longo dos últimos anos têm vindo a comprovar a utilidade e o potencial desta tecnologia para aplicações terapêuticas, particularmente na terapia génica. O potencial do sistema CRISPR/Cas9 tem vindo a progredir graças ao conhecimento cada vez mais pormenorizado dos mecanismos deste sistema e das estratégias de edição do genoma utilizadas. Existem, no entanto, ainda muitos desafios que têm de ser superados para que esta tecnologia possa ser considerada segura e eficaz no tratamento de muitas patologias.

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.