WO2022008557A2 - Modulation of cftr expression - Google Patents

Abstract

The present invention provides a DNA targeting system for modulating the expression of the CFTR gene. The present invention utilizes CRISPR/Cas9-based epigenome editing tools for repression or activation of CFTR gene expression and provides the regions of the CFTR gene that can be targeted by such system to increase the CFTR expression.

Description

MODULATION OF CFTR EXPRESSION

[001] The present invention relates to DNA targeting systems and methods utilizing such for modulating the expression of the CFTR gene.

BACKGROUND

[002] One of the major challenges in treating Cystic Fibrosis (CF) is that CFTR expression in the lung is very low. That low expression may limit the potency of approved therapeutics and may also cause some promising drugs to fail clinical trials. Specifically, all current FDA-approved compounds for treatment of CF, including Ivacaftor, Lumacaftor, Tezacaftor, and Elexacaftor physically interact with the CFTR protein to increase protein folding or ion transport activity. Increasing the amount of total CFTR protein for those drugs to target could increase their therapeutic benefit. Meanwhile, several drug candidates with promising results in engineered high CFTR-expressing cells have not been effective at physiological CFTR levels [1] and increasing CFTR expression may rescue those drugs. For those reasons, increasing CFTR expression and thus total CFTR protein in the lungs of CF patients would be a significant advance because it could improve the efficacy of already-approved CFTR drugs. Supporting this notion, a new class of drugs which aim to increase the total amount of CFTRmRNA are in development [1]

[003] The mechanisms governing CFTR gene regulation remain incompletely understood. Regulation of the CFTR locus is complex. Millions of potential gene regulatory elements have been identified across the human genome [2, 3] Differences in the activity of those regulatory elements cause genes, including CFTR, to be expressed in different patterns throughout the tissues of the body [4] Many enhancers are specifically active in one or a small number of cell types, helping to explain precise gene expression patterns. Several studies have characterized proximal CFTR enhancers and their corresponding transcription factors, revealing potential regulatory elements across diverse human cell and tissue types [5-10] Those studies create the opportunity to now develop therapeutic interventions that manipulate CFTR gene regulation for functional benefit [1, 11, 12]

[004] Due to its versatility and ease of use in screening paradigms, we here use CRISPR/Cas9 epigenome-editing proteins to characterize putative CFTR enhancer elements. Through complementary base pairing, a short guide RNA (gRNA) mediates Cas9 binding to a user-defined DNA sequence [13] By designing the 20 bp protospacer sequence that confers DNA-binding specificity, the Cas9 enzyme can be targeted to almost any region in the genome. The natural function of Cas9 is to act as a nuclease, inducing double -stranded breaks at its genomic binding site. Mutating the two endonuclease domains generates a deactivated Cas9 (dCas9) protein that has no endonuclease activity but maintains its RNA-guided DNA-binding activity [13] dCas9, in conjunction with a gRNA, functions as a modular DNA-binding protein. As such, CRISPR/dCas9 epigenome-editing proteins can modulate gene expression without making permanent changes to the underlying DNA sequence. Covalently modified histone proteins are a prominent indicator of the activity of a regulatory element [14] Acetylation of histone lysine residues, for example, is a classic mark of an active enhancer [14, 15] Fusing a histone acetyltransferase domain (p300) to dCas9 is sufficient to increase the expression of target genes more than 30 kb away in the genome [16] Similarly, fusing the Kriippel associated box (KRAB) domain to dCas9 is sufficient to recruit heterochromatin-forming proteins and repress targeted gene expression [17]

[005] Mechanisms governing the diversity of CFTR gene expression throughout the body are complex. However, it is clear that multiple intronic and distal regulatory elements play a role. This invention addresses the need for alternative ways of regulating CFTR gene expression

SUMMARY OF THE INVENTION

[006] By localizing either dCas9^p300 or dCas9^KRAB to putative CFTR regulatory elements, the present inventors identified multiple genomics regions responsible for modulating CFTR expression. Furthermore, as measured via Ussing Chamber studies in patient-derived human bronchial epithelial (HBE) cells, upregulation of mutant CFTR in combination with VX809 (Lumacaftor) treatment induced higher levels of chloride transport compared to VX809 treatment alone.

[007] The present invention provides a DNA targeting system for modulating CFTR expression, comprising a) a fusion protein comprising two heterologous polypeptide domains, wherein the first polypeptide domain comprises a Cas protein and the second polypeptide domain has transcription repression activity or transcription co-activation activity, and b) at least one guide RNA (gRNA) targeting the CFTR gene.

[008] The present invention further provides polynucleotides, vectors and compositions comprising such DNA targeting system.

BRIEF DESCRIPTION OF THE DRAWINGS

[009] The present invention is described below by reference to the following figures.

[010] Figure 1. CRISPR/dCas9 epigenome editing of CFTR enhancer elements modulates endogenous CFTR mRNA levels. (A) Locations and widths of targeted putative enhancer regions are displayed in linear genomic space and with respect to the CFTR gene structure. (B) HEK293T cells stably expression dCas9^p300 and (C) HT29 cells stably expressing dCas9^KRAB were treated with lentivirus expressing a single gRNA targeted to the putative enhancer regions of interest. Seven days post transduction, cells were harvested and evaluated for CFTR expression using qRT-PCR. Mean log fold change and standard error of the mean is shown per gRNA (* p < 0.1). With the exception of the Cas9-only control and the x-axis discontinuities between targeted regions, spacing between gRNAs is proportional to linear genomic distance and comparable across targeted regions. The mean log fold change across gRNAs for each genomic region is shown as a black bar.

[Oil] Figure 2. CRISPR/dCas9 epigenome editing of CFTR enhancer elements modulates endogenous CFTR protein levels. HEK293T cells stably expression dCas9^p300 were treated with lentivirus expressing a single gRNA targeted to either the -44kb genomic loci (A) or the promoter (B). HT29 cells stably expressing dCas9^KRAB were treated with lentivirus expressing a single gRNA targeted to the promoter (C) or the Intronl la, b genomic loci (D). Seven days post transduction, cells were harvested and evaluated for protein expression via western blot. Positive control: lOug of lysate from HEK293T transduced CFTR cDNA.

[012] Figure 3. Epigenome editing for therapeutic benefit in HBEs. D508/D508 CF donor HBEs were transduced with either a Mock (dCas9^p300 -gRNA) or with a CRISPR (dCas9^p300 +gRNA) vector. (A) Cells grown in monolyer were evaluated for CFTR expression by qRT-PCR (* p < 0.05). Samples were differentiated for 28 days in ALI culture system and evaluated for ion transport via Ussing Chamber measurements. Cultures were pre-treated with Lumacaftor (+VX809) or DMSO-only (- VX809) for 48 hours. (B) Representative trace of three replicates. Changes in short circuit current measured in response to (C) Forskolin and (D) VX770 (* p < 0.05).

DETAILED DESCRIPTION OF THE INVENTION Definitions

[013] The following definitions are used throughout the description.

[014] The term “Site-specific nuclease” as used herein refers to an enzyme capable of specifically recognizing and cleaving DNA sequences. The site-specific nuclease may be engineered. Examples of engineered site-specific nucleases include zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), and CRISPR/Cas9-based systems.

[015] The term "Transcription activator-like effector" or "TALE" as used herein refers to a protein that recognizes and binds to a particular DNA sequence. The "TALE DNA-binding domain" refers to a DNA-binding domain that includes an array of tandem 33-35 amino acid repeats, each of which specifically recognizes a single base pair of DNA. Such repeats may be arranged in any order to assemble an array that recognizes a specific sequence.

[016] The term "Transcription activator-like effector nucleases" or "TALENs" as used herein refers to fusion proteins of the catalytic domain of a nuclease, and a designed TALE DNA-binding domain that may be targeted to a custom DNA sequence.

[017] The term "Zinc finger" as used herein refers to a protein that contains a zinc finger domain and which recognizes and binds to DNA sequences.. A single zinc finger contains approximately 30 amino acids and the domain typically functions by binding 3 consecutive base pairs of DNA via interactions of a single amino acid side chain per base pair.

[018] The term "Zinc finger nuclease" or "ZFN" as used herein refers to a chimeric protein molecule comprising at least one zinc finger DNA binding domain effectively linked to at least one nuclease or part of a nuclease capable of cleaving DNA when fully transcribed and assembled.

[019] The term “CRISPR” (Clustered Regularly Interspaced Short Palindromic Repeats) refers to a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacteriophages that had previously infected the prokaryote. They are used to detect and destroy DNA from similar bacteriophages during subsequent infections.

[020] The term "CRISPR system" refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") proteins, including sequences encoding a Cas protein, a tracr (tons -activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (containing a "direct repeat" and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred herein to as a "spacer" in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus.

[021] The term “Type II CRISPR system” refers to effector system that carries out targeted DNA double-strand break in four sequential steps, using a single effector enzyme, Cas9, to cleave dsDNA. Compared to the Type I and Type III effector systems, which require multiple distinct effectors acting as a complex, the Type II effector system may function in alternative contexts such as eukaryotic cells. The Type II effector system consists of a long pre-crRNA, which is transcribed from the spacer- containing CRISPR locus, the Cas9 protein, and a tracrRNA, which is involved in pre-crRNA processing.

[022] The term “gRNA”, also used interchangeably herein as a chimeric single guide RNA (“sgRNA”), refers to nucleic acid which is a fusion of two noncoding RNAs: a crRNA and a tracrRNA.

[023] The term “Cas9” refers to an endonuclease that cleaves nucleic acid and is encoded by the CRISPR loci and is involved in the Type II CRISPR system. The Cas9 protein may be from any bacterial or archaea species, such as Streptococcus pyogenes. The Cas9 protein may be mutated so that the nuclease activity is inactivated.

[024] The term “dCas9” refers to an inactivated Cas9 protein. Examples include dCas9 from Streptococcus pyogenes with no nuclease activity. As used herein, “dCas9” refer to a Cas9 protein that has the amino acid substitutions D10A and H840A and has its nuclease activity inactivated.

[025] The term “target region”, “target sequence” or “protospacer” as used interchangeably herein refers to the region of the target gene to which the CRISPR/Cas9-based system targets.

[026] The term "complement" or "complementary" as used herein means a nucleic acid can mean Watson-Crick or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. The term "complementarity" refers to a property shared between two nucleic acid sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary.

[027] The term "promoter" as used herein means a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of same. A promoter may also comprise distal enhancer or repressor elements, which may be located as much as several thousand base pairs from the start site of transcription. A promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals.

[028] The term "enhancer" as used herein refers to non-coding DNA sequences containing multiple activator and repressor binding sites. Enhancers range from 200 bp to 1 kb in length and may be either proximal, 5' upstream to the promoter or within the first intron of the regulated gene, or distal, in introns of neighboring genes or intergenic regions far away from the locus. Through DNA looping, active enhancers contact the promoter dependently of the core DNA binding motif promoter specificity. 4 to 5 enhancers may interact with a promoter.

[029] The term “operably linked” as used herein means that expression of a gene is under the control of a promoter with which it is spatially connected. A promoter may be positioned 5' (upstream) or 3' (downstream) of a gene under its control. The distance between the promoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function.

[030] The term "vector" as used herein means a nucleic acid sequence containing an origin of replication. A vector may be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be a self- replicating extrachromosomal vector, or a DNA plasmid.

[031] The term "adeno-associated virus" or "AAV" as used interchangeably herein refers to a small virus belonging to the genus Dependovirus of the Parvoviridae family that infects humans and some other primate species. AAV is not currently known to cause disease and consequently the virus causes a very mild immune response.

[032] As used herein, the term "fusion protein" refers to a chimeric protein created through the covalent or non-co valent joining of two or more genes, directly or indirectly, that originally coded for separate proteins. In some embodiments, the translation of the fusion gene results in a single polypeptide with functional properties derived from each of the original proteins.

[033] The term "subject" and "patient" as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgous or rhesus monkey, chimpanzee, etc.) and a human). In some embodiments, the subject may be a human or a non-human. The subject or patient may be undergoing other forms of treatment.

[034] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art.

Methods for regulating the expression of a target gene

[035] Multiple methods to control gene expression have been described. Synthetic transcription factors have been engineered to control gene expression for many different medical and scientific applications in mammalian systems, including stimulating tissue regeneration, drug screening, compensating for genetic defects, activating silenced tumor suppressors, controlling stem cell differentiation, performing genetic screens, and creating synthetic gene circuits. These transcription factors can target promoters or enhancers of endogenous genes or be designed to recognize sequences orthogonal to mammalian genomes for transgene regulation. The most common strategies for engineering novel transcription factors targeted to user-defined sequences have been based on the programmable DNA-binding domains of zinc finger proteins and transcription-activator like effectors (TALEs). Both of these approaches involve applying the principles of protein-DNA interactions of these domains to engineer new proteins with unique DNA-binding specificity.

Fusion proteins

[036] The present invention relies on the use of a fusion protein for regulating CFTR gene expression, said fusion protein comprising two heterologous polypeptide domains. The first polypeptide domain comprises a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein and the second polypeptide domain has an activity selected from the group consisting of transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, nucleic acid association activity, methylase activity, and demethylase activity. The Cas protein may comprise Cas9. The Cas9 may comprise at least one amino acid mutation which knocks out nuclease activity of Cas9.

[037] The second polypeptide domain may have transcription repression activity. The second polypeptide domain may have a Kruppel associated box activity, such as a KRAB domain, ERF repressor domain activity, Mxil repressor domain activity, SID4X repressor domain activity, Mad- SID repressor domain activity or TATA box binding protein activity. More particular, the fusion protein may be dCas9^KRAB.

[038] The second polypeptide domain may have histone modification activity. The second polypeptide domain may have histone deacetylase, histone acetyltransferase, histone demethylase, or histone methyltransferase activity. The histone acetyltransferase may be p300 or CREB-binding protein (CBP) protein, or fragments thereof.

[039] The histone acetyltransferase may include a human p300 protein or a fragment thereof. The transcription co-activation domain may include a wild-type human p300 protein or a mutant human p300 protein, or fragments thereof. The transcription co-activation domain may include the core lysine -acetyltranserase domain of the human p300 protein, i.e., the p300 HAT Core (also known as "p300 WT Core"). More specifically, the fusion protein may be dCas9^p300 (such as, for example, disclosed in WO2014/197748)

Method for identifying putative regulatory elements of the CFTR gene

[040] CRISPR/Cas9-based epigenome editing provides a new and previously unexplored tool for interrogating CFTR enhancer function. The present invention demonstrates that interventions which increase the expression of CFTR may be therapeutically beneficial for the treatment of patents harboring the AF508 mutation. A better understanding CFTR regulatory mechanisms could uncover novel therapeutic interventions for the development of cystic fibrosis therapies.

[041] By targeting either dCas9^p300 or dCas9^KRAB to putative CFTR regulatory elements, the method provided by the present disclosure allows to identify multiple genomics regions responsible for modulating CFTR expression. These results demonstrate that identifying and modulating transcriptional regulatory regions of CFTR is a viable path to identifying new drug targets for the treatment of CF.

[042] The present inventors used the fusion proteins as described above to identify putative regulatory elements of the CFTR gene. The present disclosure therefore provides a method for identifying genomic regions for modulating CFTR expression, said method comprising: i. contacting target cells expressing the CFTR gene with a DNA targeting composition comprising: a) a fusion protein comprising two heterologous polypeptide domains, wherein the first polypeptide domain comprises a Cas protein and the second polypeptide domain has transcription repression activity or transcription co-activation activity, and b) at least one guide RNA (gRNA) targeting the CFTR gene; ii. measuring the change in the CFTR gene expression or CFTR protein level; and iii. identifying genomic regions where said CFTR gene expression or protein levels are increased or decreased in the presence of the DNA targeting system as genomic regions for modulating the CFTR expression.

[043] Preferably, to identify activators of CFTR expression said fusion protein comprises a polypeptide domain having transcription co-activation activity. The transcription co-activation domain may include a wild-type human p300 protein or a mutant human p300 protein, or fragments thereof. The transcription co-activation domain may include the core lysine-acetyltranserase domain of the human p300 protein, i.e., the p300 HAT Core. Preferably such fusion protein is dCas9^p300. The present disclosure provides an example (Example 1) of using such DNA targeting system to identify activators and repressors of CFTR gene expression.

Transcriptional Activators

[044] The compositions, vectors and polynucleotides of the present invention, include a nucleotide sequence encoding a transcriptional activator that activates a target gene. The transcriptional activator may be engineered. For example, an engineered transcriptional activator may be a CRISPR/Cas9- based system, a zinc finger fusion protein, or a TALE fusion protein.

[045] The CRISPR/Cas9-based DNA targeting system, as described herein, may be used to activate transcription of CFTR gene with RNA. The CRISPR/Cas9-based system may include a fusion protein, as described above, wherein the second polypeptide domain has transcription activation activity or histone modification activity. For example, the second polypeptide domain may include VP64 or p300.

[046] The transcriptional activator may be a zinc finger fusion protein. The zinc finger targeted DNA- binding domains, as described above, can be combined with a domain that has transcription activation activity or histone modification activity. For example, the domain may include VP64 or p300.

[047] TALE fusion proteins may be used to activate transcription of CFTR gene. The TALE fusion protein may include a TALE DNA-binding domain and a domain that has transcription activation activity or histone modification activity. For example, the domain may include VP64 or p300.

DNA targeting system for regulating the expression of CFTR

[048] The present invention provides to a DNA targeting system comprising: a) a fusion protein comprising two heterologous polypeptide domains, wherein the first polypeptide domain comprises a Cas protein and the second polypeptide domain has an activity selected from the group consisting of transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, nucleic acid association activity, methylase activity, and demethylase activity, and b) at least one guide RNA (gRNA), said at least one gRNA comprising a 12-22 base pair complementary polynucleotide sequence of the CFTR gene DNA sequence, followed by a protospacer-adjacent motif, wherein said at least one gRNA targets a promoter region of the CFTR gene or an enhancer region of the CFTR gene.

[049] The at least one gRNA may target an intron of the CFTR gene. The at least one gRNA may target an exon of the CFTR gene.

[050] In a particular embodiment, the present invention provides a DNA targeting system for modulating CFTR expression, comprising a) a fusion protein comprising two heterologous polypeptide domains, wherein the first polypeptide domain comprises a Cas protein and the second polypeptide domain has transcription repression activity or transcription co-activation activity, and b) at least one guide RNA (gRNA) targeting the CFTR gene.

[051] Said Cas protein is, preferably, a Cas protein lacking nuclease activity. More particularly, such protein is dCas9. More specifically, Cas9 protein has the amino acid substitutions D10A and H840A and has its nuclease activity inactivated

[052] Preferably, said fusion protein comprises a polypeptide domain having transcription co activation activity. The transcription co-activation domain may include a wild-type human p300 protein or a mutant human p300 protein, or fragments thereof. The transcription co-activation domain may include the core lysine-acetyltranserase domain of the human p300 protein, i.e., the p300 HAT Core. Preferably such fusion protein is dCas9^p300.

[053] The present invention also provides one or more isolated polynucleotides encoding said DNA targeting system. Each of the components of the DNA targeting system might be encoded by a single polynucleotide or by multiple polynucleotides. In the case of 2 polynucleotides, one of such is encoding the fusion protein and the other encoding a gRNA targeting CFTR gene. The present invention also provides one or more vectors comprising said one or more isolated polynucleotides. A single vector comprising one or more polynucleotides encoding the DNA targeting system is also provided. Alternatively, a 2-vectors system can also be used: first vector comprising a polynucleotide encoding the fusion protein and the second vector comprising a polynucleotide encoding a gRNA targeting the CFTR gene.

[054] The CRISPR/Cas9-based system could be implemented using a lentiviral vector. Such modified lentiviral vector comprises a polynucleotide sequence encoding a fusion protein and a polynucleotide sequence encoding at least one sgRNA targeting the CFTR gene. The fusion protein may be the fusion protein of the DNA-targeting system, as described above. The first polynucleotide sequence may be operably linked to a promoter. The promoter may be a constitutive promoter, an inducible promoter, a repressible promoter, or a regulatable promoter.

[055] Alternatively the DNA targeting system might be implemented using one or more AAV vectors. [056] The present invention also provides a cell comprising said isolated polynucleotide or said vector.

[057] The present invention also provides a composition for inducing CFTR gene expression in a cell. The composition comprises an isolated one or more polynucleotide sequences encoding the fusion protein and at least one guide RNA (gRNA) targeting a region of the CFTR gene. The at least one guide RNA may target a promoter region of the CFTR gene. Alternatively the at least one guide RNA may target a -44kb region of the CFTR gene. The composition may also comprise a viral delivery system. The composition may include an engineered AAV vector. The present invention also provides a cell comprising said composition for inducing CFTR gene expression in a cell.

[058] The present invention also provides a method of modulating CFTR protein levels in cells, more specifically for increasing CFTR protein levels in cells, said method comprising administering to a cell expressing a mutant CFTR gene the DNA targeting system as described above.

[059] The present invention also provides a kit comprising said composition for enhancing (more specifically, increasing) CFTR expression in a cell or said cell comprising said composition for inducing CFTR gene expression in a cell. [060] Table 1. Putative CFTR enhancers

gRNA sequences

[061] In the context of the present invention, gRNA provides the targeting of the CRISPR/Cas9-based system to the CFTR gene. The gRNA is a fusion of two noncoding RNAs: a crRNA and a tracrRNA. The gRNA may target any desired DNA sequence of CFTR gene by exchanging the sequence encoding a 20 bp protospacer which confers targeting specificity through complementary base pairing with the desired part of the CFTR gene.

[062] The DNA targeting system of the invention may include at least one gRNA, wherein the gRNAs target different DNA sequences of the CFTR gene. The target DNA sequences may be overlapping. The target sequence or protospacer is followed by a PAM sequence at the 3' end of the protospacer. Different Type II systems have differing PAM requirements. For example, the Streptococcus pyogenes Type II system uses an “NGG” sequence, where “N” can be any nucleotide.

[063] In particular, the at least one or more gRNA of the DNA targeting system targets the -44kb region of Chromosome 7 (positions 117,435,226-117,436,012 as defined in hg38) of the CFTR gene or promoter region on Chromosome 7 (positions 117,478,921-117,480,093 as defined in hg38) of the CFTR gene.

[064] In a particular embodiment, the one or more gRNAs comprise a sequence defined by SEQ ID NO 1-135. More particular, the gRNA comprises a sequence selected from the list consisting of sequences of SEQ ID NO: 1-9 and 23-42 to allow transcriptional activation of the CFTR gene (such sequences are listed in Table 2). Preferred gRNA in this case comprises the sequence of SEQ ID NO: 40.

[065] The examples of the present invention demonstrate that activating CRISPR/dCas9-based fusion protein targeted to the CFTR gene is able to modulate chloride transport in patient-derived HBEs. HBEs have been transduced with an all-in-one lentiviral vector delivering a puromycin resistance gene, dCas9^p300 fusion protein, and the most potent activating gRNA (SEQ ID NO: 40). The examples of the present invention demonstrate a significant increase in CFTR mRNA levels in such treated HBEs, confirming the effect of targeting gRNA to the specific regions of the CFTR gene.

[066] Table 2. Preferred gRNA sequences for activating CFTR expression. The start and the end locations are indicated for Chromosome 7.

Therapeutic use of the DNA targeting system

[067] The examples of the present invention demonstrate that CFTR protein levels can be modulated by using the DNA targeting system of the present invention in patient-derived HBEs, which is a representative disease model for CF.

[068] Hence, the present invention provides a method of treating CF in a subject in need thereof, the method comprising administering to the subject a DNA targeting system, vector or composition of the invention. Such DNA targeting system, vector or composition is administered in a therapeutically effective amount.

[069] The present invention also provides a DNA targeting system, vector or composition of the invention for use in the treatment of CF.

[070] The present invention also provides use of a DNA targeting system, vector or composition of the invention for the manufacture of a medicament for the treatment of CF

[071] The DNA targeting system, vectors and compositions of the present invention could be used together with other therapeutic molecules to treat patients having AF508 CFTR mutation such as, for example, VX809 and/or VX770.

Pharmaceutical compositions

[072] The composition of the present invention may be in a pharmaceutical composition. The pharmaceutical composition may comprise about 1 ng to about 10 mg of DNA encoding the CRISPR/Cas9- based system or CRISPR/Cas9-based system protein component, i.e., the fusion protein. The pharmaceutical composition may comprise about 1 ng to about 10 mg of the DNA of the modified lentiviral vector. The pharmaceutical composition may comprise about 1 ng to about 10 mg of the DNA of the modified AAV vector and a nucleotide sequence encoding the site-specific nuclease. The pharmaceutical compositions according to the present invention can be formulated according to the mode of administration to be used. In cases where pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, pyrogen free and particulate free. An isotonic formulation is preferably used. Generally, additives for isotonicity may include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstriction agent is added to the formulation.

[073] The composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be functional molecules as vehicles, adjuvants, carriers, or diluents. The pharmaceutically acceptable excipient may be a transfection facilitating agent, which may include surface active agents, such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. [074] The transfection facilitating agent can be a polyanion, polycation, including poly-L- glutamate (LGS), or lipid. The transfection facilitating agent is poly-L-glutamate, and more preferably, the poly- L-glutamate is present in the composition for genome editing in skeletal muscle or cardiac muscle at a concentration less than 6 mg/ml. The transfection facilitating agent may also include surface active agents such as immune -stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid may also be used administered in conjunction with the genetic construct. In some embodiments, the DNA vector encoding the composition may also include a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, as a DNA-liposome mixture (see for example W09324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. Preferably, the transfection facilitating agent is a polyanion, polycation, including poly-L- glutamate (LGS), or lipid.

EXAMPLES

Example 1. Identification and validation of CFTR specific enhancers

Genome accessibility data processing

[075] Normalized DNase-seq signal tracks in counts per million (CPM) were downloaded from the ENCODE project portal for the following tissues: lung, pancreas, small intestine, and transverse colon; as well as the following cell lines: A549, Caco-2, HEK293T, HT29, and SAEC)[2] For tissues and cell lines in which more than one sample was assayed, mean signal was computed using wiggletools [18]

[076] Aligned DNase-seq reads in BAM format were obtained for human epididymis epithelial (HEE) and human tracheal epithelial (HTE) cells. Read mappings were normalized to CPM and converted to a signal track using the deepTools utility bamCoverage with the following parameters: “~ ignoreDuplicates — binsize 50 — smoothLength 100 — normalizeUsing CPM” [19] Two samples were assayed for each of the primary cells, hence mean signal was computed using wiggletools [18]

Gene expression data processing

[077] The most recent tissue-specific gene expression data was downloaded from GTEx Portal (GTEx Analysis v8/dbGaP Accession phs000424.v8.p2) on Mar 25, 2020 in units of transcripts per million (TPM) [20] . Certain tissue subclassifications were collapsed into a more parsimonious set of tissues (e.g. {Amygdala,..., Substantia nigra} = Brain)

[078] Cell line and primary cell gene expression was taken from a variety of sources and expression was converted to TPM. gRNA selection

[079] Putative CFTR enhancer regions were selected by identifying regions of differential chromatin accessibility across CFTR-high vs CFTR-low cell lines and tissues (Figure 1). Regions were prioritized based on information available in the literature characterizing these sites [4-10, 21] From this analysis, 17 regions in addition to the promoter region were selected and evaluated for regulatory activity (Table 1).

[080] gRNAs were designed to span each genomic region of interest with at least 1 gRNA/ lOObp of sequence. gRNAs were optimized for lowest predicted off-target binding and highest on-target activity [22], while maintaining the desired distribution across the genomic regions of interest. This analysis resulted in design of 135 total gRNAs (Table 3).

Plasmid Construction

[081] Individual gRNAs were cloned into pLV-hU6-gRNA (Addgene plasmid #83925). Oligonucleotides for each protospacer were synthesized (IDT-DNA), hybridized, phosphorylated, and ligated into the dual BsmBI sites using conventional cloning methods. dCas9^p300 and dCas9^KRAB were expressed from pLV-EFS-dCas9^p300-T2A-Puro [23] and pLV- hUbC-dCas9^KRAB -T2A-Puro (Addgene plasmid #71236) respectively. To generate an all in one lentivirus that co expressed a gRNA along with dCas9^p300, the hU6-gRNA cassette from pLV-hU6-gRNA was cloned between the Kpnl and Pad sites of pLV-EFS-dCas9^p300-T2A- Puro.

Cell culture

[082] HEK293T cells were obtained from the American Tissue Collection Center (ATCC, Manassas, VA, USA) and were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin. HT29 cells were obtained from Sigma- Aldrich and were maintained in McCoy’s 5 A medium supplemented with 10% FBS and 1% penicillin/streptomycin. Bmil/hTERT human bronchial epithelial cell line, UNCCF3T [24], was cultured using the CRC method [25] NIH3T3J2 cells were a gift from Dr. Richard Schlegel and Dr. Xuefeng Liu at Georgetown University. Cells were cultured and irradiated as previously described [25]

Viral production and transduction of immortalized cell lines

[083] All lentiviral vectors used in this study are second generation and were produced using standard viral production methods that have been previously described [26] Briefly, 5.7 million HEK293T cells were plated per 10 cm dish. The following day, cells were transfected with lipofectamine2000 (ThermoFisher) with 10 pg of transfer vector, 3 pg of pMD2G and 8 pg psPAX2. The media was changed 12-14 hours post-transfection. The viral supernatant was collected 24 and 48 hours after this media change for a total of 20 mL of virus, and passed through a 0.45 um fdter. pLV-hU6-gRNA and pLV-hUbC-dCas9^KRAB raw viral supernatant was snap frozen. Due to the low pLV-hUbC-dCas9^p300 viral titers, the viral supernatant was concentrated to lOOx using Lenti-XTM concentrator (CloneTech) prior to being snap frozen.

[084] To generate a stable HEK293T and HT29 cell lines expressing dCas9^KRAB or dCas9^p300, cells were resuspended and plated into viral supernatant supplemented with 4 ug/ml polybrene. The viral supernatant was exchanged for fresh medium 12-24 hours later. On day 4 post transduction, cells were selected in lug/ml puromycin to obtain a pure population of transduced cells. To evaluate gRNA induced activation or repression of CFTR, HEK293T- dCas9^p300 and HT29-dCas9^KRAB cells were transduced with gRNA lentivirus using the same method. pLV-hUbC-dCas9^KRAB and pLV-U6-gRNA virus was used at lx, while pLV-hUbC- dCas9^p300 lentivirus was used at 400x due to the lower viral titer.

Flow Cytometry

[085] Cells were fixed and permeabilized using the eBioscience Foxp3/ Transcription Factor Staining Kit using the nuclear staining protocol (Thermofisher). Cells were evaluated for Cas9 expression using the a Cas9-PE conjugated antibody at 1: 100 dulution (Clone 7A9-3A3, Cell Signialling Technologies). Cells were evaluated for florescence using the Attune NxT flow cytometer (ThermoFisher).

Quantitative Reverse Transcription PCR

[086] qRT-PCR samples from HEK293T and HT29s were prepped using the Cells-to-Ct 1-Step Taqman Kit (Ambion), per the manufacturer’s instructions. mRNA from HBE cultures were isolated using the Qiagen RNeasy Plus kit. Equal mass of mRNA was reverse transcribed using Superscript VILO (ThermoFisher). Real-time PCR using multiplexed Taqman assays (CFTR: Hs0035701 l_m, TBP: Hs99999910_ml, ThermoFisher) was performed on the Quantstudio7 Detection System (ThermoFisher). The results are expressed as fold change expression of CFTR normalized to TBP using the AAC_t method.

[087] The results are expressed as fold change CFTR mRNA expression normalized to TBP expression using the AACt method. Briefly, L⁽'_t was computed as CT_,R - ⁽'rx where the reference gene ( R ) was TBP and the test gene ( X ) was CFTR. Log2 fold change, or AACT, was computed by subtracting from each well’s ACT the mean ACT for all control wells in which only Cas9 and no gRNA was added. Samples in which template was undetected were assigned CT = 40. Samples with CT values below the 95% confidence interval for mean CT — as computed per gene and per experiment (CRSPR-activation or CRSPR-inhibition) — were discarded. Reported values are the mean and SEM from two independent experiments performed on different days. One-way ANOYA was used to confirm significant effects of gRNA. Dunnetfs post hoc test was used to test for significance of effects for each gRNA, comparing the distribution of log fold changes for each gRNA to that for Cas9 only.

Western Blot Analysis

[088] Cells were lysed in RIPA buffer (Sigma-Aldrich) supplemented with protease inhibitor cocktail (Sigma- Aldrich). Protein concentration was measured using BCA protein assay reagent (ThermoFisher) and Varioskab LUX Microplate Reader (ThermoFisher). Lysates were mixed with loading buffer; equal amounts of protein were run on Mini -PROTEAN TGX 4-15% precast polyacrylamide gels (Bio-Rad) and transferred to nitrocellulose membranes using the Trans-Blot Turbo System (Bio-Rad). Nonspecific antibody binding was blocked with Intercept TBS blocking buffer (Li-Cor) for 1 h at room temperature. The membranes were incubated with the following primary antibodies: anti-CFTR Clone769 (1: 1000 dilution, University of North Carolina at Chapel Hill, Cystic Fibrosis Foundation) in Intercept T20 TBS (Li-Cor) overnight at 4 °C; anti-Actin (1:5000 dilution, Sigma-Aldrich, A2066) in Intercept T20 TBS (Li-Cor) overnight at 4 °C. The membranes were washed with TBST for 15 min and incubated for 45 min with Donkey anti-mouse 680 RD (Li- Cor, 1:5000) and Donkey anti-rabbit 800 CW (Li-Cor, 1:5000) antibodies in Intercept T20 TBS and subsequently washed with TBST for 15 min. Membranes were visualized using the Odyssey CLx (Li- Cor).

Human Bronchial Epithelial Ussing Chamber Studies

[089] UNCCF3T [24] cells were thawed from cryopreservation and cultured using the CRC method [25] UNCCF3T cells were plated in 3T3J2IRUNC-conditioned media (CM) supplemented with 5 uM rock inhibitor (Y) (Axxora) for 24 hours. Conditioned media was prepared as previously described [27] . The cells were then treated with a single lentivirus co-expressing dCas9^p300 and a CFTR targeting gRNA. Cells were transduced with a final concentration of 20x lentivirus for 3 hours in the presence of polybrene diluted 1: 1000 (Sigma TR-1003-G). Cells were then washed with PBS and fed with fresh CM + Y. Selection with 0.5 ug/mL puromycin began 6 days after transduction when the cells were -60% confluent. Cells were grown for one additional passage in CRC and seeded at a total seeding density of 1.5 x 10⁵ cells in 12-mm Millicell CM inserts (Millipore PICM01250) coated with human placental collagen (Sigma C7510) and fed UNCALI media supplemented with 0.5 ug/mL puromycin. Cells were differentiated at an air-liquid interface and treated with 5 uM VX809 for 48 hours before Ussing analysis. Ussing chamber studies were performed on day 28 with the addition of amiloride, forskolin, VX770, CFTRinh-172, and UTP as previously described [24, 25] One-way ANOVA followed by the Tukey post hoc test was used to determine statistical significance.

Results

[090] CFTR is expressed at highly variable levels throughout the body and across primary cells and cell lines. CFTR is expressed at low levels in the lung in comparison to other tissues including the pancreas, colon, salivary gland, small intestine, and epididymis. Comparing chromatin accessibility, measured by DNase-seq, around the CFTR gene between CFTR-low and CFTR-high expressing cell types reveals a substantial diversity of chromatin structure. Taken together, these data suggest CFTR expression is tightly regulated and furthermore that tissue-specific enhancers may mediate that regulation. While regulatory elements 1 Mb from the CFTR promoter are possible [28], comprehensive studies of the genetics of gene regulation suggest that most enhancers act over shorter distances [20, 29, 30] Therefore, we prioritized interrogating genomic regions within +/- 50kb of the CFTR gene. Candidate enhancers were identified by selecting genomic regions with differential chromatin structures across CFTR-low and CFTR-high expressing tissues. By integrating DNase-seq data-sets with reporter assay data [7, 8], and previously interrogated genomic regions [4-6, 10, 21], we selected 18 high-priority CFTR genomic regions of interest to interrogate further.

[091] Enhancer activity was evaluated by localizing dCas9^{KR AB} (a heterochromatin-forming, repressive transcription factor) [17] or dCas9^p300 (a euchromatin-forming, activating transcription factor) [16] to each putative enhancer region of interest and evaluating resulting changes in CFTR expression. gRNAs were designed to span each putative enhancer region with at least 1 gRNA per every 100 base pairs of sequence. gRNAs were optimized for lowest predicted off-target binding and highest on-target activity [22], while maintaining the desired distribution across the genomic regions of interest. This analysis resulted in design of 135 total gRNAs (Table 3).

[092] In order to evaluate dynamics of CFTR enhancer activity, we selected a CFTR-low (HEK293T) and a CFTR-high (HT29) cell line to evaluate enhancer-mediated modulation of CFTR expression. Stable HEK293T-dCas9^p300 and HT29-dCas9^KRAB cell lines were generated with lentivirus and validated for Cas9 expression by flow cytometry. The stable cell lines were then subsequently transduced with lentivirus expressing a gRNA of interest. Seven days post transduction, cells were harvested and evaluated for CFTR expression by qRT-PCR (Figure 1) and Western Blot (Figure 2) analysis. Significant CFTR activation was observed at the mRNA level when targeting the -44 kb and promoter regions in the CFTR-low HEK293T cells (Figure IB). Targeting a different regulatory element, Intron 1 la,b , in addition to the promoter caused significant repression of CFTR mRNA in the CFTR- high HT29 cell line (Figure 1C).

[093] We further evaluated whether changes in CFTR mRNA expression led to changes in CFTR protein expression (Figure 2). Not surprisingly, robust gene activation and repression is observed across multiple gRNAs targeting the CFTR promoter (Figure 2B,C). We were able to detect modest increases of up to 1.6 fold in CFTR protein for three of the four gRNAs targeting the -44 kb region (Figure 2A). In contrast, enhancer-mediated repression was more robust than activation in this experimental system. Repressing Intron 1 la,b with dCas9^KRAB caused 10-fold protein knockdown across the evaluated gRNAs (Figure 2D). Notably, eight enhancers were capable of reducing CFTR protein expression when repressed, despite there being no statistically significant effect on mRNA expression. The inconsistency is likely because these gRNAs induced only a moderate change at the transcript level and therefore the variability between replicates prevented statistical significance.

[094] Our data generated in model cell lines suggests that CFTR protein levels can be modulated by targeting CRISPR/dCas9 epigenome modifiers to CFTR regulator elements. Therefore, we hypothesized that increasing CFTR expression through modulation of endogenous CFTR gene regulation could enhance the effects of CFTR modulator compounds. To this end, we evaluated our most potent activating CRISPR/dCas9 epigenome complex for its ability to modulate chloride transport in patient-derived HBEs. Growth enhanced AF508/AF508 HBEs were transduced with an all-in-one lentiviral vector delivering a puromycin resistance gene, dCas9^p300, and either a polyT terminator gRNA (Mock) or our most potent activating gRNA 40 (CRISPRa). Cells were selected with puromycin to enrich for transduced cells. Before seeding air-liquid interface (ALI) cultures, a cell pellet was harvested and evaluated for CFTR expression using qRT-PCR (Figure 3a). Results showed a significant increase in CFTR mRNA levels in CRISPRa treated HBEs, thus confirming the transduction and selection protocol was successfully implemented. Puromycin selected cells were differentiated towards a mucociliary phenotype for 28 days using established methods [31]

[095] With multiple small molecule drugs already on the market for treatment of AF508/AF508 patients, any new treatments must be superior to existing options. Therefore, we evaluated if dCas9^p300 induced CFTR gene activation has any benefit over treatment with the CFTR potentiator VX770 (Ivacaftor) alone or in combination with the CFTR corrector VX809 (Lumacaftor). Mock and CRISPRa transduced cultures were pre-treated with Lumacaftor(+VX809) or DMSO-only (-VX809) for 48 hours before Ussing Chamber studies. On the day of study, cultures were sequentially treated with amiloride, forskolin, VX770, CFTRinh-172, and UTP and the subsequent changes in short circuit current were measured (Figure 3b). CRISPRa in combination with VX809 treatment resulted in a statistically significant increase in chloride transport over VX809 treatment alone (Figure 3D,F). The combined response of CRISPRa +XV809 was synergistic in nature as CRISPRa alone has little effect on chloride transport. However, CRISPRa in combination with VX770 alone showed a modest and non-significant increase in short circuit current (Figure 3E). Overall, our data shows that increased CFTR levels leads to improved ion exchange in HBE cells grown in the ALI culture system. These experiments demonstrate that interventions that increase CFTR expression may ultimately be a therapeutically beneficial adjunct treatment for individuals harboring the AF508 CFTR variant. Furthermore, CRISPR/Cas9 based epigenome modulators are a valuable tool that can be used to further elucidate the endogenous CFTR signaling mechanisms and pathways.

[096] Table 3: CFTR Targeting gRNAs. The start and end positions are indicated for Chromosome 7.

References

1. Giuliano, K.A., et al., Use of a High-Throughput Phenotypic Screening Strategy to Identify Amplifiers, a Novel Pharmacological Class of Small Molecules That Exhibit Functional Synergy with Potentiators and Correctors. SLAS Discov, 2018. 23(2): p. 111-121.

2. An integrated encyclopedia ofDNA elements in the human genome. Nature, 2012. 489(7414): p. 57-74.

3. Thurman, R.E., et al., The accessible chromatin landscape of the human genome. Nature, 2012. 489(7414): p. 75-82.

4. Bergougnoux, A., et al., A balance between activating and repressive histone modifications regulates cystic fibrosis transmembrane conductance regulator (CFTR) expression in vivo. Epigenetics, 2014. 9(7): p. 1007-17.

5. Kerschner, J.L., et al., Chromatin remodeling mediated by the FOXA1/A2 transcription factors activates CFTR expression in intestinal epithelial cells. Epigenetics, 2014. 9(4): p. 557-65.

6. Kerschner, J.L. and A. Harris, Transcriptional networks driving enhancer function in the CFTR gene. Biochem J, 2012. 446(2): p. 203-12.

7. Ott, C.J., et al., Intronic enhancers coordinate epithelial-specific looping of the active CFTR locus. Proc Natl Acad Sci U S A, 2009. 106(47): p. 19934-9.

8. Ott, C.J., et al., A complex intronic enhancer regulates expression of the CFTR gene by direct interaction with the promoter. J Cell Mol Med, 2009. 13(4): p. 680-92.

9. Yang, R., et al., Differential contribution of cis-regulatory elements to higher order chromatin structure and expression of the CFTR locus. Nucleic Acids Res, 2016. 44(7): p. 3082-94.

10. Zhang, Z., S.H. Leir, and A. Harris, Immune mediators regulate CFTR expression through a bifunctional airway-selective enhancer. Mol Cell Biol, 2013. 33(15): p. 2843-53.

11. Sasaki, S., et al., Steric Inhibition of 5' UTR Regulatory Elements Results in Upregulation of Human CFTR. Mol Ther, 2019. 27(10): p. 1749-1757.

12. Villamizar, O., et al., Targeted Activation of Cystic Fibrosis Transmembrane Conductance Regulator. Mol Ther, 2019. 27(10): p. 1737-1748.

13. Jinek, M., et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012. 337(6096): p. 816-21.

14. Zhou, V.W., A. Goren, and B.E. Bernstein, Charting histone modifications and the functional organization of mammalian genomes. Nat Rev Genet, 2011. 12(1): p. 7-18.

15. Calo, E. and J. Wysocka, Modification of enhancer chromatin: what, how, and why? Mol Cell, 2013. 49(5): p. 825-37.

16. Hilton, I.B., et al., Epigenome editing by a CRISP R-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat Biotechnol, 2015. 33(5): p. 510-7.

17. Thakore, P.I., et al., Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements. Nat Methods, 2015. 12(12): p. 1143-9.

18. Zerbino, D.R., et al., WiggleTools: parallel processing of large collections of genome-wide datasets for visualization and statistical analysis. Bioinformatics, 2014. 30(7): p. 1008-9.

19. Ramirez, F., et al., deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res, 2016. 44(W1): p. W160-5.

20. Human genomics. The Genotype -Tissue Expression (GTEx) pilot analysis: multitissue gene regulation in humans. Science, 2015. 348(6235): p. 648-60.

21. Nuthall, H.N., et al., Analysis of DNase-I-hypersensitive sites at the 3 ' end of the cystic fibrosis transmembrane conductance regulator gene (CFTR). Biochem J, 1999. 341 ( Pt 3): p. 601-11.

22. Doench, J.G., et al., Optimized sgRNA design to maximize activity and minimize off-target effects of CRISP R-Cas9. Nat Biotechnol, 2016. 34(2): p. 184-191.

23. Klann, T.S., et al., CRISPR-Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat Biotechnol, 2017. 35(6): p. 561- 568. 24. Fulcher, M.L., et al., Novel human bronchial epithelial cell lines for cystic fibrosis research. Am J Physiol Lung Cell Mol Physiol, 2009. 296(1): p. L82-91.

25. Gentzsch, M., et al., Pharmacological Rescue of Conditionally Reprogrammed Cystic Fibrosis Bronchial Epithelial Cells. Am J Respir Cell Mol Biol, 2017. 56(5): p. 568-574. 26. Salmon, P. and D. Trono, Production and titration oflentiviral vectors. Curr Protoc Neurosci,

2006. Chapter 4: p. Unit 421.

27. Liu, X., et al., Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens. Nat Protoc, 2017. 12(2): p. 439-451.

28. Claussnitzer, M., C.C. Hui, and M. Kellis, FTO Obesity Variant and Adipocyte Browning in Humans. N Engl J Med, 2016. 374(2): p. 192-3.

29. Cookson, W., et al., Mapping complex disease traits with global gene expression. Nat Rev Genet, 2009. 10(3): p. 184-94.

30. Pai, A.A., J.K. Pritchard, and Y. Gilad, The genetic and mechanistic basis for variation in gene regulation. PLoS Genet, 2015. 11(1): p. el004857. 31. Fulcher, M.L. and S.H. Randell, Human nasal and tracheo-bronchial respiratory epithelial cell culture. Methods Mol Biol, 2013. 945: p. 109-21.

32. Yoshimura, K., et al., The cystic fibrosis gene has a "housekeeping" -type promoter and is expressed at low levels in cells of epithelial origin. J Biol Chem, 1991. 266(14): p. 9140-4.

33. Montoro, D.T., et al., A revised airway epithelial hierarchy includes CFTR-expressing ionocytes. Nature, 2018. 560(7718): p. 319-324.

34. Plasschaert, L.W., et al., A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte. Nature, 2018. 560(7718): p. 377-381.

Claims

WHAT IS CLAIMED IS:

1. A DNA targeting system for modulating CFTR expression, comprising a) a fusion protein comprising two heterologous polypeptide domains, wherein the first polypeptide domain comprises a Cas protein and the second polypeptide domain has transcription repression activity or transcription co-activation activity, and b) at least one guide RNA (gRNA) targeting the CFTR gene.

2. The DNA targeting system of claim 1, wherein the second polypeptide domain has transcription co activation activity.

3. The DNA targeting system of claim 2, wherein the second polypeptide domain is wild-type human p300 protein or a mutant human p300 protein, or fragments thereof.

4. The DNA targeting system of claim 1, wherein the Cas protein is a Cas protein lacking nuclease activity.

5. The DNA targeting system of claim 1, wherein said at least one gRNA targets a promoter region of the CFTR gene or an enhancer region of the CFTR gene.

6. The DNA targeting system of claim 1, wherein the at least one guide RNA comprises a sequence selected from the list consisting of SEQ ID NOs: 1-135

7. The DNA targeting system of claim 2, wherein the at least one gRNA targets the -44kb region of the CFTR gene or the promoter of the CFTR gene.

8. The DNA targeting system of claim 7, wherein the fusion protein is dCas9^p300 and at least one gRNA comprises a sequence selected from the list consisting of SEQ ID NOs: 1-9 and 23-42.

9. One or more isolated polynucleotides encoding the DNA targeting system of any one of claims 1-8.

10. One or more vectors comprising one or more isolated polynucleotides of claim 9.

11. The one or more vectors of claim 9, wherein said vector is a lentiviral or an AAV vector.

12. A vector comprising polynucleotides encoding said DNA targeting system of any one of claims 1- 8

13. The vector of claim 12, wherein said vector is a lentiviral or an AAV vector.

14. A composition comprising the DNA targeting system of any one of claims 1-8, one or more polynucleotides of claim 9, or one or more vectors of claim 10 or 12.

15. A method for modulating CFTR protein levels in target cells, said method comprising administering to a cell having a mutant CFTR gene the DNA targeting system according to any one of claims 1-8.

16. A method of treating a subject in need thereof, having a mutant CFTR gene, the method comprising administering to the subject the DNA targeting system of any one of claims 1-8, one or more vectors of claim 10 or 12, or a composition of claim 14.

17. A pharmaceutical composition comprising the DNA targeting system of any one of claims 1-8, one or more polynucleotides of claim 9, or one or more vectors of claim 10 or 12.