<p>The growing complexity of scientific research demands further development of advanced gene regulation systems. For instance, the ultimate goal of tissue engineering is to develop constructs that functionally and morphologically resemble the native tissue they are expected to replace. This requires patterning of gene expression and control of cellular phenotype within the tissue engineered construct. In the field of synthetic biology, gene circuits are engineered to elucidate mechanisms of gene regulation and predict the behavior of more complex systems. Such systems require robust gene switches that can quickly turn gene expression on or off. Similarly, basic science requires precise genetic control to perturb genetic pathways or understand gene function. Additionally, gene therapy strives to replace or repair genes that are responsible for disease. The safety and efficacy of such therapies require control of when and where the delivered gene is expressed in vivo.</p><p>Unfortunately, these fields are limited by the lack of gene regulation systems that enable both robust and flexible cellular control. Most current gene regulation systems do not allow for the manipulation of gene expression that is spatially defined, temporally controlled, reversible, and repeatable. Rather, they provide incomplete control that forces the user to choose to control gene expression in either space or time, and whether the system will be reversible or irreversible.</p><p>The recent emergence of the field of optogenetics--the ability to control gene expression using light--has made it possible to regulate gene expression with spatial, temporal, and dynamic control. Light-inducible systems provide the tools necessary to overcome the limitations of other gene regulation systems, which can be slow, imprecise, or cumbersome to work with. However, emerging light-inducible systems require further optimization to increase their efficiency, reliability, and ease of use.</p><p>Initially, we engineered a light-inducible gene regulation system that combines zinc finger protein technology and the light-inducible interaction between Arabidopsis thaliana plant proteins GIGANTEA (GI) and the light oxygen voltage (LOV) domain of FKF1. Zinc finger proteins (ZFPs) can be engineered to target almost any DNA sequence through tandem assembly of individual zinc finger domains that recognize a specific three base-pair DNA sequence. Fusion of three different ZFPs to GI (GI-ZFP) successfully targeted the fusion protein to the specific DNA target sequence of the ZFP. Due to the interaction between GI and LOV, co-expression of GI-ZFP with a fusion protein consisting of LOV fused to three copies of the VP16 transactivation domain (LOV-VP16) enabled blue-light dependent recruitment of LOV-VP16 to the ZFP target sequence. We showed that placement of three to nine copies of a ZFP target sequence upstream of a luciferase or eGFP transgene enabled expression of the transgene in response to blue-light. Gene activation was both reversible and tunable based on duration of light exposure, illumination intensity, and the number of ZFP binding sites upstream of the transgene. Gene expression could also be spatially patterned by illuminating the cell culture through photomasks containing various patterns.</p><p>Although this system was useful for controlling the expression of a transgene, for many applications it is useful to control the expression of a gene in its natural chromosomal position. Therefore we capitalized on recent advances in programmed gene activation to engineer an optogenetic tool that could easily be targeted to new, endogenous DNA sequences without re-engineering the light inducible proteins. This approach took advantage of CRISPR/Cas9 technology, which uses a gene-specific guide RNA (gRNA) to facilitate Cas9 targeting and binding to a desired sequence, and the light-inducible heterodimerizers CRY2 and CIB1 from Arabidopsis thaliana to engineer a light-activated CRISPR/Cas9 effector (LACE) system. We fused the full-length (FL) CRY2 to the transcriptional activator VP64 (CRY2FL-VP64) and the N-terminal fragment of CIB1 to the N-, C-, or N- and C- terminus of a catalytically inactive Cas9. When CRY2-VP64 and one of the CIBN/dCas9 fusion proteins are expressed with a gRNA, the CIBN/dCas9 fusion protein localizes to the gRNA target. In the presence of blue light, CRY2FL binds to CIBN, which translocates CRY2FL-VP64 to the gene target and activates transcription. Unlike other optogenetic systems, the LACE system can be targeted to new endogenous loci by solely manipulating the specificity of the gRNA without having to re-engineer the light-inducible proteins. We achieved light-dependent activation of the IL1RN, HBG1/2, or ASCL1 genes by delivery of the LACE system and four gene-specific gRNAs per promoter region. For some gene targets, we achieved equivalent activation levels to cells that were transfected with the same gRNAs and the synthetic transcription factor dCas9-VP64. Gene activation was also shown to be reversible and repeatable through modulation of the duration of blue light exposure, and spatial patterning of gene expression was achieved using an eGFP reporter and a photomask. </p><p>Finally, we engineered a light-activated genetic "on" switch (LAGOS) that provides permanent gene expression in response to an initial dose of blue light illumination. LAGOS is a lentiviral vector that expresses a transgene only upon Cre recombinase-mediated DNA recombination. We showed that this vector, when used in conjunction with a light-inducible Cre recombinase system,1 could be used to express MyoD or the synthetic transcription factor VP64-MyoD2 in response to light in multiple mammalian cell lines, including primary mouse embryonic fibroblasts. We achieved light-mediated upregulation of downstream myogenic markers myogenin, desmin, troponin T, and myosin heavy chains I and II as well as fusion of C3H10T½ cells into myotubes that resembled a skeletal muscle cell phenotype. We also demonstrated LAGOS functionality in vivo by engineering the vector to express human VEGF165 and human ANG1 in response to light. HEK 293T cells stably expressing the LAGOS vector and transiently expressing the light-inducible Cre recombinase proteins were implanted into mouse dorsal window chambers. Mice that were illuminated with blue light had increased microvessel density compared to mice that were not illuminated. Analysis of human VEGF and human ANG1 levels by enzyme-linked immunosorbent assay (ELISA) revealed statistically higher levels of VEGF and ANG1 in illuminated mice compared to non-illuminated mice.</p><p>In summary, the objective of this work was to engineer robust light-inducible gene regulation systems that can control genes and cellular fate in a spatial and temporal manner. These studies combine the rapid advances in gene targeting and activation technology with natural light-inducible plant protein interactions. Collectively, this thesis presents several optogenetic systems that are expected to facilitate the development of multicellular cell and tissue constructs for use in tissue engineering, synthetic biology, gene therapy, and basic science both in vitro and in vivo.</p> / Dissertation

Cell migration is an important component of many physiological and pathological processes such as tissue and organ morphogenesis during development, wound healing, inflammatory immune response, and tumor metastasis. The actin cytoskeleton is the basic engine driving cell migration. In the present study, we elucidate the role of an important actin interacting proteins, Adducins, in motility of normal lung epithelium and lung cancer cells. Adducins are the family of cytoskeleton protein capping the fast growing end and facilitating the bundling of actin filaments. Adducins are encoded by the three closely related genes namely alpha (ADD1), beta (ADD2) and gamma (ADD3) Adducin. ADD1 and ADD3 are ubiquitously expressed, whereas ADD2 is most abundant in brain and erythrocytes. Adducins are also involved in recruiting spectrin to the actin filaments forming spectrin-actin membrane skeletal network. Its role in cell motility remains controversial. In this study, we observed that CRISPR/Cas9 mediated stable knockout of ADD1 and ADD3 in 16HBE normal lung epithelium cells significantly increases transfilter migration of cells. On the other hand, stable overexpression of ADD1 in H1299 Non-Small Cell lung cancer cells significantly decreases wound healing, transfilter migration and Matrigel invasion of the cells. Importantly, the effects of Adducin depletion and overexpression on cell motility were not due to altered cell proliferation. ADD1 overexpressed H1299 cells were characterized by the increased adhesion and spreading on the collagen matrix. Fluorescence microscopy revealed alterations in their cortical actin cytoskeleton that was manifested in the assembly of peripheral F-actin bundles and formation of filopodia-like protrusions. These findings suggest that Adducins are negative regulators of motility of normal lung epithelial and lung cancer cells that act by altering the architecture of submembranous actin cytoskeleton and modulating cell adhesion to the extracellular matrix.

No description available.

Hepatitis B virus (HBV) represents one of the hot topics of current basic and pharmaceutical research. Although an effective vaccine against HBV exists since 1982, the world prevalence of chronic infection is still alarming. The infection can lead to significant liver damage, often resulting in hepatocellular carcinoma. Chronic HBV infection cannot be cured due to the fact that the viral genome persists in the infected hepatocyte hidden from the host immune response as well as from the antiviral treatment. One of the novel approaches aiming for HBV cure suggests that this cccDNA pool could be destroyed using gene editing tools such as CRISPR/Cas9 system. In order to shift this gene editing system to possible medicinal application, CRISPR/Cas9 has to be specifically delivered into the target cell in order to minimize its putative off-target activity. This thesis focuses at first on the design and efficacy testing of new sgRNAs targeting HBV cccDNA and secondly, it describes modular lipid nanoparticles developed specially for delivery of the CRISPR/Cas9 system in the form of RNA. Keywords: hepatitis B virus, CRISPR/Cas9, gene editing, lipid nanoparticles, mRNA delivery, targeted delivery

Oxidative phosphorylation apparatus (OXPHOS) is responsible for production of majority of ATP in mammalian organisms. This process, occurring in the inner mitochondrial membrane, is partly regulated by nuclear-encoded subunits of cytochrome c oxidase (COX), the terminal enzyme of electron transport chain. Cox4 subunit, participating in OXPHOS regulation, is an early-assembly state subunit, which is necessary for incorporation of Cox2 catalytic subunit, thus for assembly of catalytically functional COX enzyme. Moreover, regulated expression of two isoforms (Cox4i1, Cox4i2) of Cox4 subunit is hypothesized to optimize respiratory chain function according to tissue oxygen supply. However, the functional impact of the isoform switch for mammalian tissues and cells is still only partly understood. In the present thesis, unique HEK293 cell line-based model with complete absence of subunit Cox4 (knock-out, KO) was prepared employing novel CRISPR CAS9-10A paired nickase technology and further characterized. Knock-out of both isoforms Cox4i1 and Cox4i2 (COX4i1/4i2 KO clones) showed general decrease of majority of Cox subunits resulting in total absence of fully assembled COX. Moreover, detected Complex I subunits as well as the content of assembled Complex I were decreased in COX4i1/4i2 KO clones. On the...

Att förstå olika tumörers biologi är en viktig förutsättning för att kunna utveckla nya cancerbehandlingsmetoder. Ett nytt verktyg inom cancerterapin, både för att förstå tumörers uppkomst samt hitta nya läkemedelsmål och behandlingar, är det mycket potenta genredigeringsverktyget Clustered Regularly Interspaced Short Palindromic Repeats med CRISPR-Associerade proteiner, CRISPR-Cas9. Det är ett adaptivt immunförsvar funnet hos prokaryoter. CRISPR är ett programmerbart RNA-guidat system som har DNA som mål. Tekniken går att tillämpa inom cancerimmunologin genom att t ex manipulera T-celler på olika sätt. Syftet med den här litteraturstudien var att: 1) undersöka vilka idag pågående kliniska prövningar, med cancerimmunologisk inriktning, som använder sig av CRISPR-Cas9 samt 2) vilka resultat som framkommit; 3) hur CRISPR-Cas9 ska kunna levereras till celler in vivo; 4) vilka problem har stötts på samt 5) hur framtiden kan se ut med CRISPR inom cancerforskningen. Information inhämtades under tidsperioden januari till maj 2019 på främst PubMed, clinicaltrials.gov och Google. Idag pågår 8 kliniska prövningar men studierna har ännu inte publicerat några resultat. Att finna en lämplig leveransmetod för leverans av CRISPR till målcellen är en av de stora utmaningarna med CRISPR där virala metoder är den leveransmetod som hittills har använts mest. Många studier undersöker möjligheterna med lipida nanobärare men ingen leveransmetod överträffar någon annan i dagsläget. Problem som framkommit med CRISPR-Cas9-tekniken är att metoden kan orsaka cancer vid redigering i celler som saknar p53 (ett viktigt tumör-supressor-protein); det kan orsaka patogena konsekvenser pga långa deletioner då Cas9 klyvt DNA; samt Cas9 kan klyva på andra ställen i genomet än det önskade. I framtiden kan CRISPR, med olika Cas-proteiner, komma att användas för att bland annat tillverka universella T-celler med chimär antigenreceptor (CAR-T-celler), förstå tumörers uppkomst och utveckling, finna nya läkemedelsmål, radera DNA-sekvenser från virus som inkorporerat sitt genom i vårt, och kanske även för redigering i könscellslinjen för att minska risker för bland annat cancerutveckling. / Understanding the biologi of tumors is an important prerequisite to develop new methods for cancer treatment. The gene editing tool Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) with CRISPR-associated proteins (Cas9), CRISPR-Cas9 is a new tool in cancer therapy, both for understanding the origins of the tumors and for finding treatments and drug targets. It is an adaptive immune system in prokaryotes. CRISPR is a programmable RNA- guided system which targets DNA. The technology is applicable in cancer immunology by e.g. manipulating T-cells. The purpose of this literature study was to: 1) investigate which cancer immunotherapy clinical trials, using CRISPR-Cas9, that are ongoing today and 2) which results have emerged so far; 3) how CRISPR-Cas9 can be delivered to cells in vivo; 4) which problems have arisen, and 5) what the future might hold for CRISPR within cancer research. The information was gathered from January to May 2019 from primarily PubMed, clinicaltrials.gov, and Google. Today, 8 clinical trials are ongoing but the studies have not yet published any results. One of the main challenges of CRISPR is finding a suitable methodology of delivery where viral methods is the one that has mainly been used. Many studies investigate possibility of lipid nanocarriers but as of today, no one single delivery system is superior to the others. The problems with CRISPR-Cas9 that have emerged is that it can cause cancer when editing cells that are missing p53 (an important tumor suppressing protein); it has pathogenic consequenses due to long deletions as Cas9 cuts DNA; and Cas9 can cut the DNA at off-targeted sites. In the future, CRISPR with different Cas-proteins, can be used to manufacture universal Chimeric antigen receptor T cells (CAR-T-cells), understanding the origin and development of tumors, finding new drug targets, deleting DNA-sequences from viruses that have incorporated their genome into ours, and maybe also for editing the germ line in order to reduce the risk of e.g. developing cancer.

N-glycosylated proteins that traverse the endoplasmic reticulum (ER) can make use of the calnexin cycle to attain their correct fold. The calnexin cycle modifies the N-glycan structure and allows for association of the glycoprotein with the ER lectins calnexin and calreticulin, which in turn recruit further chaperones that assist folding. Most enveloped viruses encode glycoproteins, which, upon infection of a host cell, crucially depend on the calnexin cycle to aid their folding. This includes diverse families such as Flaviviridae, Retroviridae and Orthomyxoviridae. We studied the calnexin cycle components with the ultimate aim of developing broad-spectrum antivirals. X-ray crystallography was used to structurally characterise the murine ER α-glucosidase I, which controls entry into the calnexin cycle, with a number of inhibitory antiviral iminosugars. These data reveal flexibility in the ligands' alkyl tails and may act as a basis for the discovery of enzyme specific inhibitors. UDP-glucose: glycoprotein glucosyltransferase (UGGT) is the quality control checkpoint of the calnexin cycle whose full-length structure from the thermophilic fungus Chaetomium thermophilum was recently determined. Presented here are a higher resolution structure in addition to SAXS studies of UGGT's interaction with Sep15, a protein that enhances UGGT activity. UGGT's reaction releases into the ER lumen UDP, which is the only known small molecule inhibitor of UGGT. An ER-resident UDPase, ENTPD5, breaks down UDP into UMP. Enzymatic characterisation of ENTPD5 reveals its substrate specificities; in addition we show a paralog, ENTPD6, possesses similar activities. Presented here is work towards crystallisation of these two proteins and a test of the anti-Zika activity of ENTPD5 inhibitors. Finally, CRISPR/Cas9 knock-out cells were generated to test, in principle, whether modulation of the activity of proteins involved in the calnexin cycle could be antiviral. The data confirm that the ER glucosidases are likely the best targets of those studied.

The serine/arginine-rich (SR-) family proteins constitute a diverse group of pre-mRNA splicing factors that are essential for viability. They can be characterised based on the presence of one or two RRMs and an RS domain. A subset, of which SRSF1 is the prototype, is capable of nucleocytoplasmic shuttling; a process governed by continual cyclic phosphorylation of the RS domain. In contrast, SRSF2, another member of the SR family, is unable to shuttle due to the presence of a nuclear retention sequence (NRS) at the C-terminus of its RS domain. When this NRS is fused to SRSF1, it prevents nucleocytoplasmic shuttling of the SRSF1-NRS fusion protein. In addition to its nuclear roles, SRSF1 is directly associated with the translation machinery and can activate mRNA translation of target transcripts via an mTOR-dependent mechanism. The specific mRNA translational targets that SRSF1 serves to regulate encode numerous factors including RNA processing factors and cell-cycle proteins. The aim of this work is to study the physiological relevance of SRSF1 cytoplasmic functions, as previous data have relied on overexpression systems. CRISPR/Cas9 editing was used to knock-in the NRS naturally present in SRSF2 at the SRSF1 genomic locus, creating an SRSF1-NRS fusion protein. After numerous attempts, it was only possible to obtain a single viable homozygous clone in mouse embryonic stem cells (mESCs), despite being able to successfully tag the genomic SRSF1 locus. This strongly suggests that the ablation of SRSF1 shuttling ability is highly selected against in mESCs. To assess the physiological importance of SRSF1 nucleocytoplasmic shuttling during development, a mouse model for SRSF1-NRS was also developed. SRSF1-NRS homozygous mice are born at correct Mendelian ratios, but are small in size and present with severe hydrocephalus. Finally, proteomics was used to identify interactors of endogenous cytoplasmic SRSF1 and those that bind the NRS of SRSF2 to gain insights into the mechanism of nuclear retention for non-shuttling SR proteins. In summary, this work analyses the physiological relevance of cytoplasmic SRSF1 function and the consequences of the SRSF1-NRS allele in mouse development.

Mosaic, activating mutations in PIK3CA, the gene encoding the catalytic p110α subunit of class IA phosphatidylinositol 3-kinase (PI3K), are the cause of rare, developmental growth disorders collectively known as PIK3CA-Related Overgrowth Spectrum (PROS). Given the pressing need for targeted therapy and evidence for tissue- and cell lineage-specific distribution of PIK3CA mutations in PROS, developmental models of this disease will be a key asset for preclinical drug testing and for a better understanding of PIK3CA activation in development. This PhD project addressed the lack of human, developmental PROS models by establishing isogenic series of human induced pluripotent stem cells (iPSCs) with endogenously expressed, activating PIK3CA mutations. This involved the optimisation of a CRISPR/Cas9 protocol for efficient knockin of different PIK3CA variants into human iPSCs. An isogenic iPSC series was established with cells expressing either wild-type PIK3CA or PIK3CA-H1047R, knocked into either one or both endogenous alleles. In parallel, mosaic patient- derived fibroblast cultures were reprogrammed to obtain isogenic wild-type and heterozygous iPSCs expressing PIK3CA-E418K. The models were used in comprehensive signalling studies, providing new insights into PI3K signalling in human iPSCs and how it is perturbed by genetic p110α activation. PIK3CA-E418K, a rare variant in both PROS and cancer, caused minimal pathway activation, in contrast to the highly recurrent variant PIK3CA-H1047R which induced strong PI3K signalling in both heterozygous and homozygous iPSCs according to a graded pattern. Studies of clinically relevant PI3K pathway inhibitors provided proof-of-concept that stem cell-based PROS models can be used for preclinical drug testing, and demonstrated that p110α is likely to be the main catalytic isoform mediating canonical PI3K signalling in human iPSCs. Differentiation assays revealed allele dose-dependent effects of PIK3CA-H1047R on stemness, with homozygous iPSCs exhibiting widespread transcriptome remodelling affect- ing genes implicated in cancer and development. Accordingly, these cells showed increased expression of pluripotency genes such as NANOG and NODAL, resulting in self-sustained "stemness" in embryoid body and teratoma assays. In comparison, heterozygous mutants behaved similar to wild-type controls under all differentiation paradigms. Furthermore, evidence was obtained that strong activation of PI3K signalling is fully compatible with definitive endoderm formation, arguing against cell-autonomous differentiation defects as the cause of endoderm sparing in PROS. In summary, these studies demonstrate the utility of human stem cell-based models of PROS for preclinical drug testing and for improved understanding of class IA PI3K signalling in human development. They are also likely to be useful in efforts to obtain a better understanding of PIK3CA-H1047R in human cancer.

A mucopolissacaridose tipo I (MPS I) é causada pela deficiência de alfa-L-iduronidase (IDUA), responsável pelo catabolismo de glicosaminoglicanos (GAGs), levando ao acúmulo multissistêmico de sulfato de heparano e dermatano. Este estudo tem por objetivo avaliar o potencial de sistemas lipídicos nanoestruturados como carreadores do plasmídeo do sistema CRISPR/Cas9 e um vetor doador da sequência do gene IDUA/Idua para edição gênica em fibroblastos de pacientes e em modelo murino de MPS I. Foram produzidos lipossomas (DOTAP, DOPE e DSPE-PEG) e nanoemulsões (e TCM) por homogeneização à alta pressão e microfluidização. O DNA foi associado às formulações por adsorção, ou por encapsulamento dos complexos pré-formados DNA/DOTAP no núcleo oleoso da nanoemulsão. A eficiência de transfecção dos complexos foi avaliada em fibroblastos de pacientes MPS I e ocorreu um aumento significativo da atividade de IDUA em 2, 15 e 30 dias após os tratamentos, que promoveu uma redução na quantidade de lisossomos nos fibroblastos tratados. A caracterização físico-química de formulações produzidas por microfluidização complexadas a somente um plasmídeo ou juntamente com um oligonucleotídeo foi verificada e pode-se afirmar que a capacidade de complexação e transfecção depende diretamente do tipo celular e da relação de cargas, e não há implicações quanto ao tamanho das sequências de ácidos nucleicos. Camundongos MPS I receberam os complexos lipossomais por injeção hidrodinâmica e sua biodistribuição foi detectada principalmente no pulmão, coração e fígado. A atividade sérica de IDUA normal aumentou em cerca de 6% e foi mantida por seis meses. A atividade aumentada no pulmão, coração, fígado e rim após eutanásia promoveu redução dos GAGs na urina e nos mesmos tecidos, corroborando com as análises histológicas. Em um estudo em andamento, foi realizada uma investigação mais aprofundada do efeito do tratamento lipossomal na morfologia óssea, sistemas cardiovascular e respiratório, e funções cerebrais dos animais tratados. A análise ecocardiográfica demonstrou uma melhora na hipertrofia e contratilidade do coração, porém não houve melhora na espessura das válvulas. O diâmetro da aorta foi similar ao de animais normais, porém as quebras de elastina ficaram entre o grupo normal e o não tratado. A morfologia facial dos animais tratados foi intermediária, assim como a espessura do osso zigomático. Entretanto, o osso femoral demonstrou espessura comparável ao normal. Já a resistência pulmonar apresentou uma tendência de redução nos animais tratados em relação aos animais MPS I. O conjunto de resultados demonstra o potencial das nanoestruturas lipídicas co-complexadas com o plasmídeo CRISPR/Cas9 e um vetor doador da sequência IDUA/Idua para terapia gênica da MPS I. / Mucopolysaccharidosis type I (MPS I) is caused by the deficiency of alpha-L-iduronidase (IDUA), responsible for the catabolism of glycosaminoglycans (GAGs), leading to multisystemic accumulation of heparan and dermatan sulfate. This study aims to evaluate the potential of lipid-based nanostructures as carriers of the CRISPR/Cas9 plasmid and a vector donor of the IDUA/Idua sequence for gene editing in patients’ fibroblasts and in a murine model of MPS I. Liposomes (DOTAP, DOPE, and DSPE-PEG) and nanoemulsions (also MCT) were produced through high-pressure homogenization or microfluidization. DNA was associated with liposomes and nanoemulsions by adsorption or by encapsulation of DNA/DOTAP preformed complexes in the oil core of nanoemulsions. The transfection efficiency of complexes was evaluated in fibroblasts from MPS I patients and a significant increase in IDUA activity was demonstrated at 2, 15, and 30 days after treatments. It was also possible to observe a significant reduction in lysosomal amount in treated fibroblasts. The physicochemical characterization of liposomes and nanoemulsions produced through microfluidization complexed with a single plasmid or along with an oligonucleotide has been verified and it can be stated that the complexing and transfection capacity of the complexes depends directly on the cell type and the charge ratio, and there are no implications of the size of the nucleic acid sequences. MPS I mice received the liposomal complexes by hydrodynamic injection and their immediate biodistribution was detected mainly in the lung, heart, and liver. An increase of about 6% in normal serum IDUA activity was maintained for six months, in addition to increased lung, heart, liver, and kidney activity after euthanasia. The enhanced enzymatic activity promoted a significant GAGs reduction in urine and in the same tissues, corroborating with histological analysis. In an ongoing study, a deeper investigation was carried out on the effect of liposomal treatment on bone morphology, cardiovascular and respiratory systems, and brain function. The echocardiographic analysis showed an improvement in the parameters of hypertrophy and contractility of the heart, but there was no improvement in heart valves. Aorta diameter was similar to that of normal animals, but elastin breaks were between the normal and untreated groups. Facial morphology of treated animals was intermediate, as well as the analysis of zygomatic bone thickness. However, femoral bone showed thickness comparable to normal animals. Lung resistance, on the other hand, showed a tendency to reduction in treated animals when compared to MPS I. The set of results demonstrates the potential of the co-complexed lipid nanostructures with the CRISPR/Cas9 plasmid and a donor vector of the IDUA/Idua sequence for MPS I gene therapy.