Polynucleotide construct for gene editing

Abstract

The present application relates to a polynucleotide construct for gene editing suitable to hybridize with a CRISPR-Cas9 ribonucleoprotein complex. The disclosed polynucleotide construct is suitable for the spatially controlled release of CRISPR-Cas9 ribonucleoprotein for gene editing. The gene editing potential of the construct in a composition was demonstrated in vitro at single cell level. The safety and gene editing of the construct was also demonstrated. This construct offers a new strategy for the delivery of the CRISPR system with spatial control.

Description

[0001] POLYNUCLEOTIDE CONSTRUCT FOR GENE EDITING

[0002] Technical field

[0003] The present application relates to polynucleotide construct for gene editing suitable to hybridize with a CRISPR-Cas9 ribonucleoprotein complex. The disclosed construct is suitable for the controlled release of CRISPR-Cas9 ribonucleoprotein complex for gene editing.

[0004] Background art

[0005] Genome editing using clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9)[1] hashigh potential for the treatment of neurological diseases originated from genetic mutations including Alzheimer's disease,[2]Huntington disease,[3]amyotrophic lateral sclerosis,[4]among others. Many of the advances made in CRISPR brain delivery systems have been deployed taking advantage of viral vectors, more specifically, adeno- associated virus (AAV) . However, AAVs have a low packing capacity, require specific infrastructures for the large scale production, and pose safety concerns due to their immunogenicity[5]and integration into the DNA breaks created in Cas9 breaks.[6]Non-viral CRISPR / Cas delivery systems have emerged as an alternative to viral delivery approaches. Non- viral formulations have been used in the context of adult brain[7] using different delivery cargos including plasmid DNA encoding Cas9 and sgRNA,[8]Cas9 mRNA plus sgRNA[9]and Cas9 ribonucleoprotein (Cas9 complexed with sgRNA) ,[7c] being the last two more desirable for clinical translation. These non-viral vectors, administered by intracerebral routes, have allowed successful gene editing in different brain cells.[7a'7b'7e'10]One of the limitations of current non- viral vectors is their limited spatial resolution. Nanoparticles (NPs) once injected can diffuse from the injection site into the brain parenchyma depending on their morphology and chemistry.

[0011] The spatial control over gene editing requires light-activatable NPs,

[0012] and in particular, near-infrared (NIR) light-activatable NPs to increase tissue penetration (millimeter range1131) . NIR- activatable CRISPR formulations have been reported for gene editing outside the brain, in particular tumor models,[8a'141using Cas9 / sgRNA encoding plasmids[8'151and Cas9 / sgRNA ribonucleoprotein complex (RNP) ,[14' 16] pu|-nop inside the brain. These formulations showed important limitations such as: (i) limited gene editing efficiency (<10% of the cells) ; [st] (ii) high laser power requirements to release the gene editing system, compromising cell viability;[14b](iii) leaching of the formulation in the absence of NIR activation;[15'171(iv) composition by lanthanides that may induce toxicity.

[0016] In addition, brain application of NIR- activatable CRISPR formulations raise significant challenges, considering that: (i) the laser should penetrate the skull and activate brain cells transfected with the NPs,

[0006] (ii) the triggering system should not activate gliosis and,

[0007] (iii) NP activation should display spatial resolution.

[0008] Previous work includes 'Modulation of Angiogenic Activity by Light- Activatable miRNA-Loaded Nanocarriers ' and 'Intracellular delivery of more than one protein with spatiotemporal control' by M. Lino et al.. However, none of these documents disclose a polynucleotide construct such as the present invention.

[0009] Summary The present invention relates to a polynucleotide construct for gene editing suitable to hybridi ze with a CRISPR-Cas 9 ribonucleoprotein complex, wherein the construct comprises :

[0010] - a first region comprising an anchor group suitable to immobili ze the polynucleotide construct in a substrate ;

[0011] - a second region comprising a spacer sequence;

[0012] - a third region comprising a ssDNA sequence suitable to hybridi ze with the sgRNA sequence of a sgRNA: Cas 9 protein complex, forming a ssDNA: sgDNA: Cas 9 complex .

[0013] In one embodiment the anchor group is positioned at the 3 ' end of the polynucleotide construct .

[0014] In one embodiment the anchor group is positioned at the at the 5 ' end of the polynucleotide construct .

[0015] In one embodiment the substrate is selected from a nanocarrier, gold nanorods , polymeric nanoparticles , nanocrystals , plane substrates , medical devices , polymeric substrates such as polyurethane or silicone substates , metal substrates such as gold or titanium substrates , nanocomposite substrates , ceramic substrates , or plane substrates modi fied with nanocarriers .

[0016] In one embodiment the nanocarrier has an average si ze between 5 and 500 nm .

[0017] In one embodiment the anchor group is selected from a thiohexyl group, a N-hydroxysuccinimide group, a vinyl group, an alkyne group, a cycloalkane group, an epoxide group, an azide group, an amine group, a pyrrole group, or a carboxyl group . In one embodiment the spacer sequence is positioned at the 3 ' end of the polynucleotide construct .

[0018] In one embodiment the spacer sequence is positioned at the 5 ' end of the polynucleotide construct .

[0019] In one embodiment the spacer sequence is a sequence of the same nucleotide , a polypeptide , a synthetic monomer sequence that is a substitute for amino acids , or an oligomer such as a divinyl oligomer .

[0020] In one embodiment the spacer sequence comprises between 16 and 34 thymine nucleotides .

[0021] In one embodiment the ssDNA sequence of the third region is designed as a nucleotide sequence complementary to , and suitable to , hybridi ze with the sgRNA sequence selected with at least 70% of hybridi zation .

[0022] In one embodiment the ssDNA sequence of the third region is selected according to its melting temperature between 40 and 70 ° C .

[0023] In one embodiment it comprises a fourth region comprising a nucleotide sequence suitable for enzymatic cleavage .

[0024] In one embodiment the polynucleotide construct is for use in the treatment of neurological diseases originated from genetic mutations including Al zheimer' s disease , Huntington disease , amyotrophic lateral sclerosis .

[0025] General description

[0026] Here is disclosed a polynucleotide construct for gene editing suitable to hybridi ze with a CRI SPR-Cas 9 ribonucleoprotein complex. This construct is suitable for the controlled release of CRISPR-Cas9 ribonucleoprotein complex for gene editing .

[0027] The CRISPR-Cas9 complex / system comprises a single guide RNA (sgRNA) and Cas9 nuclease, which together form a ribonucleoprotein (RNP) complex.

[0028] The delivery of Cas9 protein rather than Cas9 DNA was selected to prevent immunogenic responses.1181The construct of the present invention takes advantage of a ssDNA polynucleotide sequence that can be immobilized in a substrate, such as nanocarriers like gold nanorods (AuNRs) , to immobilize the complex sgRNA: Cas9 (RNP) . In this example, the core formed by the nanocarrier can induce dehybridization mechanisms of the ssDNA immobilized in the surface of the nanocarrier with complementary sequence of the sgRNA (Figure la) .

[0029] The construct was rationally designed in terms of nucleotide sequence, spacer and composition to: (i) minimize leaching of the ribonucleoprotein complex in the absence of activation / release, (ii) prevent the cleavage by the Cas9 system (Figure lb) and (iii) avoid the denaturation of the Cas9 protein.

[0030] The gene editing properties of this construct was evaluated in vitro using brain cells and in vivo following intracerebral or intranasal administration in a reporter transgenic mouse. Brief description of drawings

[0031] For easier understanding of this application, figures are attached in the annex that represent the preferred forms of implementation which nevertheless are not intended to limit the technique disclosed herein.

[0032] Figure 1- Tn vitro delivery of Cas9-RNP mediated efficient genome editing. (a) Schematic representation of the synthesis of gene editing construct with a nanocarrier, (b) Oligonucleotide sequences used for the immobilization in the AuNR. (c) Representative structures of the Cas9 system with different spacer lengths. The representations on the top panel correspond to cluster structures from the simulated systems. The sgRNA non-hybridized (top right of the protein) ; the sgRNA bases that are hybridized to the ssDNA (inside of Cas9 protein) ; and the poly ( thymine ) spacer (at the bottom right of the protein) . (d) Schematic representation of NIR light-controlled release of RNP for gene editing. The HeLa- d2eGFP were incubated with AuNR- dsDNA- RNP conjugates for 4 h, washed, fresh medium was added, and cells were irradiated for 3 min with a NIR laser. After 72 h, eGFP knockout was monitored by a high-content fluorescence microscope. (e) Percentage of GFP-knockout cells. RNAiMAX lipof ectamine was used as a positive control. The eGFP fluorescence normalized to the non-treated cells. Data are expressed as mean ± SEM (n = 2-6) . One-way ANOVA with post hoc Tukey' s multiple comparisons test was performed: (****) , p < 0.0001. (f) Allele frequency in HeLa cells edited with a AuNR-dsDNA ( 16T ) - RNP construct.

[0033] Figure 2- Gene editing of mouse brain cells by light- activable AuNR-dsDNA-RNP . (a) Schematic representation for the gene editing of brain cells. The excision of a stop codon by the designer of two sgRNA sequences will lead to a production of tdTomato fluorescence reporter protein. Cells were transfected with AuNR-dsDNA ( 32T ) -RNP for 4 h, washed, feed with fresh medium and then irradiated for 3 min by a NIR laser with a power of 700 mW. After 72 h, tdTomato fluorescence was monitored by flow cytometry analyses, (b- f ) Representative flow cytometry scatter plots of SVZ (b) , astrocytes- (d) and neurons-enriched cell populations (f) treated with AuNR-dsDNA32T-RNP with and without NIR light, (c-g) Percentage of tdTomato positive cells in SVZ (c) , astrocytes- (e) and neurons-enriched populations (g) as assessed by flow cytometry. Positive cells were gated based in the basal expression of tdTomato fluorescence in nontreated cells. In (c) , (e) and (g) the results are Mean ± SEM (n=3 independent experiments; 2 replicates per independent experiment) . ***, **** denote statistical significance (p < 0.001; p < 0.0001) assessed by one-way ANOVA followed by a Tukey' s post-hoc test.

[0034] Figure 3- In vivo expression of tdTomato in Ai9 adult mice trigger by light-activable AuNR-dsDNA ( 32 T) -RNP. (a) Schematic representation of stereotaxic injection of AuNR- dsDNA ( 32T ) -RNP in adult Ai9 mice, (b) AuNR accumulation in the brain of mice after intracerebral administration. Quantification was done by ICP-MS in brains at days 0 and 15 after the injection. Results are Mean ± SEM (n=3 animals) . Unpaired two-sample t-test was performed: (*) , p < 0.05. (c) Representative fluorescence images of tdTomato-positive cells in the irradiated and non-irradiated brain ventricles after the AuNR injection and in the saline injection and NIR irradiation. Scale bar is 300 pm. (d) Fold change of tdTomato fluorescence after NIR light activation. Results are Mean ± SEM (n=3-6 animals, one section per animal) . Unpaired two- sample t-test was performed: (***) , p < 0.001. (e) Distribution of tdTomato-positive cells. Results are Mean ± SEM (n=4-5 animals, 3-6 imagens per marker) . *, ** denote statistical significance (p < 0.05; p < 0.01) assessed by one-way ANOVA followed by a Tukey' s post-hoc test. (f) Experimental set up of intranasal administration. Adult Ai9 mice were administered intranasally with (i) AuNR- dsDNA ( 32T ) -RNP or (ii) PBS for 3 days. Each day, 1 h after intranasal administration, part of the animal skull was irradiated while the other part was protected with aluminum foil to prevent laser activation, (g) Quantification of AuNR- dsDNA ( 32T ) -RNP in the brain after a single intranasal administration. Animal was sacrificed 4 h post administration. The gold amount was determined by ICP-MS in the nasal cavity, olfactory bulb (OB) , cortex and hippocampus. Results are expressed as % ID (initial dose of Au) per gram of tissue. Mean ± SEM (n=4 animals for AuNR and 2 animal for saline group) . (h) Representative image of the tdTomato expression in irradiated and non-irradiated OB two weeks after the multiple nasal administrations. Scale bars = 50 pm. GL : glomerular layer, EPL: external plexiform layer, (i) Quantification of tdTomato fluorescence. The NIR-exposed (+NIR) side was normalized to the non-exposed side of the OB (-NIR) . Results are expressed as Mean ± SEM (n= 4 animals treated with AuNR-dsDNA ( 32T ) -RNP and n=3 with PBS; 20 to 24 images were obtained from each animal) . Statistical analysis was performed by a two-tailed unpaired t test, with * denoting statistical significance (P = 0.0176) .

[0035] Detailed description of embodiments

[0036] Now, preferred embodiments of the present application will be described in detail with reference to the annexed drawings. However, they are not intended to limit the scope of this application.

[0037] The polynucleotide construct comprises three regions:

[0038] - a first region comprising an anchor group suitable to immobilize the polynucleotide construct to a substrate;

[0039] - a second region comprising a spacer sequence;

[0040] - a third region comprising a ssDNA sequence suitable to hybridize with the sgRNA sequence of a sgRNA:Cas9 protein complex, forming a ssDNA: sgDNA: Cas9 complex.

[0041] In the context of the present invention, the ssDNA: sgDNA: Cas9 complex is also known as dsDNA:RNP complex.

[0042] In one embodiment, the anchor group is positioned at the 3' end of the polynucleotide construct. In another embodiment, the anchor group is positioned at the at the 5' end of the polynucleotide construct.

[0043] In one embodiment, the substrate is selected from, but not limited to, a nanocarrier, gold nanorods, polymeric nanoparticles, nanocrystals, among other nanocarriers. In one embodiment, the nanocarriers have an average size range between 5 to 500 nm. In another embodiment, the substrate can also be selected from plane substrates or medical devices from, but not limited to, polymeric substrates such as polyurethane or silicone substates, metal substrates such as gold or titanium substrates, nanocomposite substrates, ceramic substrates, among others that one skilled in the art might identify. In another embodiment, the plane substrates are modified with nanocarriers. In one embodiment , the anchor group is selected from, but not limited to , a thiohexyl group, a N-hydroxysuccinimide group, a vinyl group, an alkyne group, a cycloalkane group, an epoxide group, an azide group, an amine group, a pyrrole group, or a carboxyl group .

[0044] The thiohexyl group reduces steric interactions with the end of the polynucleotide construct and the surface of substrate .

[0045] The anchor group is selected according to the substrate and should be suitable to immobili ze the polynucleotide construct sequence to the selected substrate .

[0046] In one embodiment , the spacer sequence is positioned at the 3 ' end of the polynucleotide construct . In one embodiment , the spacer sequence is positioned at the 5 ' end of the polynucleotide construct .

[0047] In a preferred embodiment , the spacer sequence is a ssDNA sequence of the same nucleotides . In one embodiment , the spacer sequence can also be selected from, but not limited to , a polypeptide , a synthetic monomer sequence that is a substitute for amino acids , or an oligomer such as a divinyl oligomer .

[0048] The length of the spacer sequence spacer must be one suitable to repel the substrate , so the polynucleotide construct doesn' t adhere to the substrate while also allowing the ssDNA: sgDNA: Cas 9 complex to be exposed . The length of the spacer sequence influences the hybridi zation percentage between the ssDNA of the third region and the sgRNA. The spacer sequence should have a suitable length as to allow enough space between the substrate and the sgRNA: Cas 9 complex in order to allow correct hybridization of the ssDNA sequence of the third region with the sgRNA sequence in the enzyme pocket .

[0049] In one embodiment, the spacer sequence comprises 16, 24, 32 or 34 nucleotides.

[0050] In one embodiment, the spacer sequence comprises between 16 and 34 thymine nucleotides.

[0051] In one embodiment, the ssDNA sequence of the third region is designed as a nucleotide sequence complementary to, and suitable to, hybridize with the sgRNA sequence selected. The percentage of hybridization between ssDNA: sgRNA is at least 70% .

[0052] In one embodiment, both the ssDNA sequence and the spacer sequence should be of an enough length to prevent cleavage of the ssDNA by spCas9 since the enzyme may cleave the ssDNA even in the absence of a protospacer adjacent motif (known as PAM sequence) .

[0053] The sgRNA sequence is variable and selected to match the desired target gene intended to be edited by the CRISPR-Cas9 complex .

[0054] In one embodiment, the hybridized zone between ssDNA: sgRNA is at least between 5 and 30 nucleotides.

[0055] In one embodiment, the melting temperature of the hybridized zone is between 40 and 70°C. In one embodiment, the ssDNA sequence of the third region is selected according to its melting temperature between 40 and 70 °C.

[0056] The release of the sgRNA:Cas9 complex from the construct can occur via different approaches.

[0057] In one embodiment, a suitable substrate such as a nanocarrier that is a AuNR, or any other nanocarrier sensitive to near infrared (NIR) light, may absorb NIR light and convert it into heat, which leads to the release of the sgRNA:Cas9 complex from the rest of the construct.

[0058] In one embodiment, the polynucleotide construct may comprise a fourth region comprising a nucleotide sequence suitable for enzymatic cleavage, for example via protease action. This cleavable nucleotide sequence may be part of the spacer sequence. In one embodiment, the cleavable sequence comprises between 3 and 20 nucleotides.

[0059] In one embodiment, the polynucleotide construct may comprise a number of nucleotides with a melting point suitable for the release of the sgRNA:Cas9 complex from the construct upon the application of heat.

[0060] The polynucleotide construct of the present invention is suitable for use in the treatment of neurological diseases originated from genetic mutations including, but not limited to, Alzheimer's disease, Huntington disease, amyotrophic lateral sclerosis, among others; through the local delivery of the constructs in the cell niche of the interest. The present invention also relates to a composition comprising the polynucleotide construct .

[0061] Example

[0062] Herein is described one example of the application of the present invention, selecting AuNR as the substrate and NIR light as release method . The following example is not restrictive of the entire scope of the invention and serves merely as at least one proof of concept of the invention .

[0063] For the purpose of this example , polynucleotide constructs were prepared with the sequences of Table 1 and of Table 2 .

[0064] The polynucleotide construct sequence was selected from the list disclosed in Table 1 , but not limited to :

[0065] *a thiohexyl group anchor is included for each sequence for sake of example .

[0066] Table 2 . Sequence list of constructs used in in vi tro gene editing of brain cells . *a thiohexyl group is included for each sequence for sake of example.

[0067] Table 3: Sequence list of sgRNAs used to constructs design.

[0068] To immobilize the RNP to the AuNR, a ssDNA sequence of 20 bases was selected (SEQ ID NO 1 to 4) , complementary to the sgRNA (SEQ ID NO 9) , modified at the 3' or 5' terminal with different poly ( thymine ) spacers (8, 16 and 32) and containing a terminal thiol group (thiohexyl group) to allow covalent attachment to the AuNR surface (Figure lb) . The poly ( thymine ) spacer was used to distance the oligonucleotide hybridization sequence from the AuNR surface. The length of the spacers was inspired by molecular modeling studies (see below) . As a control, a sequence of 20 bases without complementarity to the sgRNA (NC) (SEQ ID NO 5) was used. The AuNRs had an average length of 42 ± 0.3 nm and width of 13 ± 0.2 nm. The number of ssDNA per AuNR was on average 403 ± 28, with a coupling efficiency of ssDNA to AuNR of nearly 40%. Next, it was evaluated whether the ssDNA immobilized in the surface of AuNRs were accessible for hybridization with sgRNA. With the exception of the non-complementary (NC) ssDNA, the sgRNA was able to hybridize with all AuNR-ssDNA constructs .

[0069] Thus, the interaction of the sgRNA to AuNRs seems to be mediated by nucleotide complementarity and not by other interactions. To evaluate the release properties of the construct, the AuNR-ssDNA was hybridized with the sgRNA and then irradiated by a NIR laser. The activation of AuNR- dsDNA: sgRNA by a NIR laser with a power of 1000 mW increased the temperature o f the suspension from 21 ° C to 41 ° C leading to the release of approximately 60% of the immobilized sgRNA from the construct in 10 min . Next , the AuNRs-ssDNA constructs were exposed to the RNP for 1 h at room temperature (between 25 and 30 ° C ) to allow the hybridi zation of the RNP to the complementary sequence of the ssDNA immobili zed in the AuNR . The immobili zation ef ficiency was slightly higher (more than 60% ) for the constructs containing ssDNA with 16 or 32 thymines than with 8 thymines . The immobili zation of the RNPs in the AuNR-ssDNA constructs altered the zeta potential from -40 mV to -20 mV . The construct that results from the hybridi zation of the RNPs with the oligonucleotides immobili zed in AuNR-dsDNAnT-RNP was relatively stable for 24 h in water as no signi ficant changes were observed in the absorbance spectrum . Importantly, the Cas 9 enzyme was not able to cleave the double stranded DNA that resulted from the hybridi zation between the target sequence of sgRNA with the ssDNA.

[0070] To investigate the interaction of the sgRNA with the ssDNA Molecular Dynamics (MD) analyses were performed . Because the ssDNA is covalently attached to the AuNR, the interaction of the 20 bases ssDNA to the sgRNA is conditioned by the length of the poly ( thymine ) spacer . The spacer needs to be long enough for the 2 Ont ssDNA to reach the complementary sgRNA strand in the enzyme pocket .[ 1N Moreover, both ssDNA and spacer length should be rationally designed to prevent the cleavage of the ssDNA by spCas 9 since the enzyme may cleave the ssDNA even in the absence of a protospacer adj acent moti f ( known as PAM sequence ) J201The distance of the enzyme from one side to the other is around 80 A, assuming an interphosphate distances of ssDNA that can range between 5 . 9 A and 7 A depending on the sugar puckering,[2'21]a 8T spacer will just cover 52 A, which is roughly half of the protein, meaning that, with that spacer, only a few bases can reach the complementary sgRNA (Figure Ic-i) . For the longer spacers (16T and 32T) it was modeled possible hybridization levels between ssDNA and sgRNA. According to the simulations for the complex with ssDNA-16T, for 6 bases of the spacer to remain outside the protein, only 14 out of 20 bases would reach the sgRNA for hybridization (Figure Ic-ii) . With the longer spacer (ssDNA-32T) 24 bases pairs of the spacer were accessible to the solvent if a hybridization of 14 bases of the ssDNA occurred. It was also modeled the fully hybridized complex with ssDNA-32T (Figure Ic-iii) . In this case 11 bases were accessible to the solvent. The MD simulations showed that the protein equilibrates in a relatively fast way. Overall, the length of the 8T spacer was not enough for the immobilization of the RNP to the AuNR; however, both 16T and 32T spacers allow the hybridization of the ssDNA with the sgRNA in the enzyme pocket.

[0071] Next, the release of the RNP from the AuNR construct was evaluated after NIR irradiation. Initially, it was evaluated the thermostability of Cas9 enzyme after NIR activation. It has been shown that Cas9 protein loses its stability to temperatures above 45°C.

[0022] Soluble Cas9 enzyme was incubated with AuNRs (without ssDNA conjugation) , exposed to a NIR laser at different powers for 3 min and then centrifuged to collect the supernatant for protein quantification and activity. NIR laser powers above 800 mW / cm2lead to the denaturation and subsequent precipitation of the enzyme. Yet, Cas9 collected from a colloidal suspension of AuNRs activated at 700 mW / cm2(or even at 1000 mW / cm2) and transfected with sgRNA and RNAiMAX in d2eGFP-HeLa cells showed activity.

[0072] Thus, for the release studies of RNP from AuNRs a NIR laser at 700 mW / cm2for 3 min was used. In these conditions, approximately 31.5% of the RNP is released from the construct .

[0073] To evaluate the cellular uptake of AuNR-dsDNA-RNP, HeLa cells were transfected with each construct (SEQ ID NO 1 to 5) with different spacers (8T, 16T and 32T) (50 pg / mL) for 4 h, and then the concentration of gold within cells was measured by inductive coupled plasma mass spectrometry (ICP-MS) . The results showed that the different constructs had different levels of cellular uptake, being the AuNR-dsDNA ( 16T ) -RNP construct the one with the highest internalization likely favoured by its aggregation and subsequent sedimentation in cell culture medium. To evaluate the intracellular trafficking of the constructs, Cas9 was labelled with DyLight 650 (DL) before the formation of the complex with the sgRNA. The AuNR-dsDNA ( 16T) -RNP-DL were incubated with HeLa cells and activated with a NIR laser (780 nm, power: 700 mW / cm2, 3 min) . The intracellular trafficking of AuNR-dsDNA-RNP was characterized by confocal microscopy after staining the endolysosomal compartment with Lysotracker Red. The results indicate that the exposure of the cells to NIR contributed for the endolysosomal escape of the nanocarrier. TEM analyses showed that the exposure of cells to NIR led to an increase in the number of disrupted endolysosomes. To further determine whether AuNR-dsDNA-RNP induced endolysosomal compartment disruption, galectin-9 was monitored by immunofluorescence, a membrane damage sensor that has been used to demonstrate endosomal escape of therapeutic molecules .

[0023] Hydroxychloroquine, which has been shown to induce endolysosomal membrane damage

[0023] , has been used as positive control. Indeed, the cells exposed to AuNR- dsDNA ( 32T ) -RNP and activated by NIR laser showed an increased number of galectin-9 foci compared to those without laser activation suggested the disruption of the endolysosomal compartment .

[0074] Overall, the results indicated that the level of cellular internalization of AuNRs-dsDNA-RNP depends in the length of ssDNA and that the escape from endolyosomal compartment was enhanced by the activation of the construct by NIR light.

[0075] To evaluate the gene editing potential of the constructs, the GFP fluorescence in d2eGFP-HeLa cells was knockout by the different AuNR-dsDNA-RNP constructs (SEQ ID NO 1 to 5) . For this purpose, cells were transfected with each construct (50 pg / mL) for 4 h in cell culture medium, either irradiated or not at 780 nm (700 mW / cm2) for 3 min and finally cultured for 3 days (Figure Id) . Cellular viability and GFP knockout were evaluated by high-content microscopy. Cells transfected with AuNR-dsDNAl 6T-RNP (SEQ ID NO 2) showed a decrease in number after light activation indicating some cytotoxicity of the construct. The remaining constructs had negligible effect in cell viability. Cells treated with the constructs but without light activation showed low levels of eGFP knockout activity, specifically below 30% in the AuNR- dsDNA32T-RNP construct (SEQ ID NO 3) (Figure le) . Importantly, cells transfected with AuNR-dsDNAl 6T-RNP (SEQ ID NO 2) or AuNR-dsDNA32T-RNP (SEQ ID NO 3) and activated by light showed eGFP knockout comparable to the one observed in cells transfected with lipof ectamine RNAiMAX complexed with

[0076] RNP, used as a positive control (Figure le) . The gene editing was further confirmed by Illumina NGS analyses, the indels (~64% ) were observed around the intended cleavage site (Figure If) further confirming the cellular gene editing. As expected, the edited cells showed low eGFP expression for several passages demonstrating the biological effect over time. The gene editing can be initiated by NIR laser activation immediately after cell transfection (4h after exposure to the construct) or 20 h after cell transfection, and thus the biological process can be controlled in time. Furthermore, the AuNR construct enabled gene editing with single-cell resolution when confocal microscopy was used for NIR activation.

[0077] Importantly, AuNRs with a complementary sequence of the sgRNA was critical for the immobilization of the RNP to the AuNR- ssDNA system. Constructs containing ssDNA that were not complementary to the terminal sequence (SEQ ID NO 5) of the sgRNA or that were inverted (SEQ ID NO 6) (the thymines were included in the 5' and not in the 3' terminal) were not active. Overall, the results showed the capacity to gene editing cells with spatio-temporal resolution. Considering the cell viability and gene editing results, the construct AuNR-dsDNA32T-RNP (SEQ ID NO 3) was selected for further analyses .

[0078] To demonstrate in vitro the gene editing of brain cells, subventricular zone (SVZ) , astrocytes and neuronal-enriched cells were isolated from Rosa26-tdTomato transgenic Ai9 mice. This mouse has a cassette inserted into the Gt (ROSA) 26Sor locus with a LoxP-Stop-LoxP-tdTomato sequence. Two sgRNA sequences (sgRNA-loxPl and sgRNA-loxP2) (SEQ ID NO 10 and 11) were designed for the deletion of LoxP-Stop cassette and expression of tdTomato fluorescent protein (Figure 2a) . AuNR with RNP-loxPl and with RNP-loxP2 were prepared separately to allow maximal control in the composition of each construct. The SVZ cells, astrocytes and neurons were incubated with AuNR-dsDNA ( 32T ) -RNP (equal amounts of AuNR-RNP-loxPl (SEQ ID NO 7) and AuNR-RNP- loxP2 (SEQ ID NO 8) for 4 h, exposed or not to a NIR laser and then cultured for 1 day (cytotoxicity assay) or 3 days (gene editing assay; Figure 2a) . Brain cells transfected with AuNR-dsDNA ( 32T ) -RNP showed no significant cytotoxicity at 24 h after irradiation. In the case of SVZ cells the irradiation was performed 24 h after the AuNR incubation because after the 4 h of incubation most of the construct was adsorbed in cell membrane and not internalized. The gene editing efficiency was determined by the quantification of tdTomato-positive cells at day 3 post-transfection by flow cytometry. The percentage of tdTomato-expressing cells in SVZ cells, astrocytes and neurons was 8%, 30% and 10%, respectively (Figures 2b-2g) . The gene editing efficiency was higher in astrocytes than SVZ cells likely because the first ones internalized higher concentration of AuNR- dsDNA ( 32T ) -RNP than the second ones. In the SVZ cells, most of the edited cells were immature (Nestin+) . Yet, the highest percentage of gene editing occurred in astrocytes (GFAP+ cells) and neuronal-like cells (NeuN+cells) . The gene editing in the SVZ cells was further confirmed by Illumina NGS analyses, the indels (~6.4%) were observed around the intended cleavage site confirming the cellular gene editing. Altogether, the results showed the capacity to gene editing brain cells with efficacy similar to the one observed for the commercial transfection agent RNAiMAX; however, with spatial resolution. To demonstrate in vivo the gene editing properties of the construct the NIR laser penetration was initially evaluated. It was determined that a 780 nm laser at 1000 mW / cm2power delivered through the brain calvaria bone (thickness: 3 mm) had an attenuation of 60% of the initial power, which was sufficient to knock out eGFP expression up to 50% in d2GFP HeLa cells and 25% in astrocytes cells. To demonstrate in vivo the gene editing of brain cells, AuNR-dsDNA ( 32T ) -RNP was stereotaxically injected into the sub-ventricular zone of both hemispheres of adult Ai9 brain mice, and just one of the brain hemispheres was exposed to NIR light (Figure 3a) . The expression of tdTomato was evaluated two weeks later by confocal microcopy. During this period, the concentration of AuNR in the brain decreased from 12 (this means 0.3 mg of NRs per Kg of animal which is much below the brain toxicity of these nanomaterials1241) to 5 ,g of Au per g of brain tissue, as confirmed by ICP-MS analyses (Figure 3b) . In other organs, such as spleen and liver, the concentration of AuNR show an increase at day 5, as confirmed by ICP-MS analyses. The results show that the expression of tdTomato fluorescence was higher (~ 4-fold) in irradiated versus non-irradiated hemisphere (Figure 3c, d) . The tdTomato positive cells were found in immature / progenitor (Sox2+; 38%) , astrocytes (GFAP+; ~40%) , microglia (IBA1+; 10%) and neuronal progenitor cells (NeuN+; 15%) (Figures 3e) . Importantly, it was not observed measurable microglia activation in the region exposed to the NIR laser that undergo gene editing in vivo.

[0079] In a separate experiment, the possibility of administering AuNR-RNP by a non-invasive route was investigated, specifically, by intranasal administration (Figure 3f-3i) . The results show that most of the AuNR accumulated in the nasal cavity, followed by the olfactory bulb (Figure 3g) . Importantly, gene editing was observed in the irradiated brain hemisphere particularly in the olfactory bulb. Here, the expression of tdTomato fluorescence was higher in the irradiated (~ 4-fold) as compared to the non-irradiated hemisphere (Figures 3h and 3i) .

[0080] In conclusion, a gene-editing construct was developed for on-demand release into deep tissues with spatial resolution. This technology offers new possibilities to perform brain gene editing with increased safety.

[0081] This description is of course not in any way restricted to the forms of implementation presented herein and any person with an average knowledge of the area can provide many possibilities for modification thereof without departing from the general idea as defined by the claims. The preferred forms of implementation described above can obviously be combined with each other. The following claims further define the preferred forms of implementation.

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Claims

CLAIMS1. A polynucleotide construct for gene editing suitable to hybridize with a CRISPR-Cas9 ribonucleoprotein complex, wherein the construct comprises:- a first region comprising an anchor group suitable to immobilize the polynucleotide construct in a substrate;- a second region comprising a spacer sequence;- a third region comprising a ssDNA sequence suitable to hybridize with the sgRNA sequence of a sgRNA:Cas9 protein complex, forming a dsDNA: sgDNA: Cas9 complex.

2. The polynucleotide construct according to the previous claim, wherein the anchor group is positioned at the 3' end of the polynucleotide construct.

3. The polynucleotide construct according to claim 1, wherein the anchor group is positioned at the at the 5' end of the polynucleotide construct.

4. The polynucleotide construct according to any of the previous claims, wherein the substrate is selected from a nanocarrier, gold nanorods, polymeric nanoparticles, nanocrystals, plane substrates, medical devices, polymeric substrates such as polyurethane or silicone substates, metal substrates such as gold or titanium substrates, nanocomposite substrates, ceramic substrates, or plane substrates modified with nanocarriers.

5. The polynucleotide construct according to any of the previous claims, wherein the nanocarrier has an average size between 5 and 500 nm.6 . The polynucleotide construct according to any of the previous claims , wherein the anchor group is selected from a thiohexyl group, a N-hydroxysuccinimide group, a vinyl group, an alkyne group, a cycloalkane group, an epoxide group, an azide group, an amine group, a pyrrole group, or a carboxyl group .7 . The polynucleotide construct according to any of the previous claims , wherein the spacer sequence is positioned at the 3 ' end of the polynucleotide construct .8 . The polynucleotide construct according to any of the claims 1 to 7 claims , wherein the spacer sequence is positioned at the 5 ' end of the polynucleotide construct .9 . The polynucleotide construct according to any of the previous claims , wherein the spacer sequence is a sequence of the same nucleotide , a polypeptide , a synthetic monomer sequence that is a substitute for amino acids , or an oligomer such as a divinyl oligomer .10 . The polynucleotide construct according to any of the previous claims , wherein the spacer sequence comprises between 16 and 34 thymine nucleotides .11 . The polynucleotide construct according to any of the previous claims , wherein the ssDNA sequence of the third region is designed as a nucleotide sequence complementary to , and suitable to , hybridi ze with the sgRNA sequence selected with at least 70% of hybridi zation .12 . The polynucleotide construct according to any of the previous claims , wherein the ssDNA sequence of the thirdregion is selected according to its melting temperature between 40 and 70 ° C .13 . The polynucleotide construct according to any of the previous claims , wherein it comprises a fourth region comprising a nucleotide sequence suitable for enzymatic cleavage .14 . The polynucleotide construct according to any of the claims for use in the treatment of neurological diseases originated from genetic mutations including Al zheimer' s disease , Huntington disease , amyotrophic lateral sclerosis .