Systems, methods, and compositions for de-repressing pkd1

Abstract

Provided herein is a system for inhibiting a miRNA-17 family miRNA from binding to the 3'UTR of PKD1, where the system includes: a gRNA; and a polynucleotide-programmable nucleotide-binding domain, where the system modifies a binding site of a miRNA-17 family miRNA in the 3'UTR of PKD1, thereby preventing binding of the miRNA-17 family miRNA and de-repressing PKD1 mRNA.

Description

SYSTEMS, METHODS, AND COMPOSITIONS FOR DE-REPRESSING PKD1CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Patent Application Serial No. 63 / 472,794, filed 06 / 13 / 2023 and incorporated by reference in its entirety.GOVERNMENT SUPPORT

[0002] This invention is supported, in whole or in part, under Federal Grant Number 1R41DK138689-01, awarded by the National Institutes of Health. The Federal Government may have certain rights in this invention.BACKGROUND

[0003] Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic disorder in the world and is caused by mutations in the polycystin-1 (PKD1) or polycystin-2 (PKD2) genes. These PKD1 or PKD2 mutations cause cyst formation in renal tubular epithelial cells that bilaterally enlarge the kidney and lead to kidney failure in adulthood. There is no cure for ADPKD and only one FDA-approved therapy (tolvaptan). Tolvaptan is a vasopressin 2 receptor antagonist and has several side effects that limit its life-long, daily use such as frequent urination and liver injury. Moreover, neither tolvaptan, nor other smallmolecule therapies in clinical trials can reverse or prevent ADPKD. Utilizing gene therapy to restore functional levels of PKD1 or PKD2 offers a promising new treatment avenue for ADPKD that could serve as an outright cure by preventing cyst formation altogether.

[0004] Many small molecule therapies for ADPKD have failed in clinical trials either due to toxicity or lack of efficacy (i.e., venglustat, bardoxolone, tesevatinib, everolimus, lixivaptan). As a chronic disease, ADPKD requires frequent re-dosing throughout the patient’s lifetime, which makes it difficult to avoid long-term toxicity at dosages that are also efficacious. Gene therapy avoids this long-term toxicity issue because it is curative after only a single (or small number) of doses. The use of CRISPR-Cas editing to correct individual ADPKD patient mutations is one possible gene therapy approach; however, this requires customizing the therapy for each patient family since most families carry different PKD 1 or PKD2 mutations. There is an urgent need fora one-size-fits-all gene therapy approach for ADPKD to provide an outright cure for this serious, life-threatening disease.SUMMARY

[0005] Provided herein is system for inhibiting an miRNA-17 family miRNA (e.g., miR-17) from binding to the 3’ UTR of PKD1, the system comprising; a gRNA; and a polynucleotide- programmable nucleotide-binding domain, where, upon introducing the system into a cell, the system genetically modifies a binding site of a miRNA-17 family miRNA in the 3’ UTR of PKD1, thereby preventing binding of the miRNA-17 family miRNA and de-repressing PKD1 mRNA.

[0006] In one aspect, this disclosure features a guide RNA (gRNA) comprising: a spacer sequence complementary to a genomic site in 3’ UTR of PKD1; and a scaffold capable of associating with a polynucleotide-programmable nucleotide-binding domain and guiding the polynucleotide-programmable nucleotide-binding domain to the genomic site in the 3’ UTR of PKD1.

[0007] In one aspect, this disclosure features a guide RNA (gRNA) comprising: a spacer sequence complementary to at least 17, 18, 19, or 20 contiguous nucleotides of the sequence of SEQ ID NO; 11; and a scaffold capable of associating with a polynucleotide-programmable nucleotide-binding domain and guiding the polynucleotide-programmable nucleotide-binding domain to a sequence complementary to the spacer sequence.

[0008] In one aspect, this disclosure features a guide RNA (gRNA) comprising: a spacer sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO; 1-10, and SEQ ID NO: 12-20, 244, 245; and a scaffold capable of associating with a polynucleotide- programmable nucleotide-binding domain and guiding the polynucleotide-programmable nucleotide-binding domain to a sequence complementary to the spacer sequence.

[0009] In some embodiments of the gRNA, the gRNA further comprises the components of a prime editing guide RNA.

[0010] In some embodiments of the gRNA, the gRNA further comprises one or more modified nucleotides.

[0011] In some embodiments of the gRNA, the gRNA is a synthetic gRNA.

[0012] In some embodiments of the gRNA, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 8.

[0013] In some embodiments of the gRNA, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO:3.

[0014] In some embodiments of the gRNA, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 2.

[0015] In one aspect, this disclosure features a system for inhibiting a miRNA-17 family miRNA from binding to the 3’ UTR of PKD1, the system comprising: any of gRNA described herein; and a polynucleotide-programmable nucleotide-binding domain or a polynucleotide encoding the polynucleotide programmable nucleotide-binding domain.

[0016] In some embodiments of the system, upon introducing the system into a cell, the system genetically modifies a binding site of a miRNA-17 family miRNA in the 3’ UTR of PKD1, thereby preventing binding of the miRNA-17 family miRNA and de-repressing PKD1 mRNA.

[0017] In some embodiments of the system, the polynucleotide-programmable nucleotide- binding domain is selected from: a base editor, a prime editor, a Cas9, a Cas9 nickase, a dead Cas9, a Casl2a, a Casl2a nickase, a dead Casl2a, a FokI nuclease, an integrase, a zinc finger nuclease, or a transcription activator-like effector nuclease.

[0018] In some embodiments of the system, the polynucleotide-programmable nucleotide- binding domain is a base editor.

[0019] In some embodiments of the system, the base editor is a cytosine base editor (CBEs).

[0020] In some embodiments of the system, the base editor is an adenine base editor (ABEs).

[0021] In some embodiments of the system, the base editor is a non-canonical CBE base editor or a non-canonical ABE base editor.

[0022] In some embodiments of the system, the polynucleotide-programmable nucleotide- binding domain is a Streptococcus pyogenes Cas 9 (SpCas9) or a Staphylococcus aureus (SaCas9).

[0023] In some embodiments of the system, (i) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 1 and the polynucleotide-programmable nucleotide- binding domain is VQR-BE3; (ii) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 2 and the polynucleotide-programmable nucleotide-binding domain is selected from: ABEmax -VRQR, NG-ABEmax, ABEmax, xABEmax, xCas9(3.6)- ABE, and xCas9(3.7)-ABE; (iii) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 3 and the polynucleotide-programmable nucleotide-binding domain is SpCas9; (iv) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 5 and the polynucleotide-programmable nucleotide-binding domain is selected from: CP-CBEmax, NG-ABEmax, and CP-ABEmax; (v) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 7 and the polynucleotide- programmable nucleotide-binding domain is selected from: VRQR, NG, xBE, xCas9(3.6)-ABE, xCas9(3.6)-CBE, xCas9(3.7)-ABE, and xCas9(3.7)-CBE; (vi) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 8 and the polynucleotide- programmable nucleotide-binding domain is selected from BE4max, AncBE4max, ABE8e, ABEmax, ABE8e-CP1028, ABE8e-NG, ABEmax-NG, CBE6a, CBE6b, CBE6c, CBE6d, ABE8e-spRY; (vii) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 9 and the polynucleotide-programmable nucleotide-binding domain is all non-canonical ABE / CBE editors; (viii) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 10 and the polynucleotide-programmable nucleotide- binding domain is SaCas9 or SaCas9-KKH; (ix) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 12 and the polynucleotide-programmable nucleotide-binding domain is SpCas9; (x) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 13 and the polynucleotide-programmable nucleotide- binding domain is SpCas9; (xi) the gRNA comprises a spacer sequence having at least 80%sequence identity to SEQ ID NO: 14 and the polynucleotide-programmable nucleotide-binding domain is SpCas9; (xii) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 15 and the polynucleotide-programmable nucleotide-binding domain is SaCas9 or SaCas9-KKH; the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 16 and the polynucleotide-programmable nucleotide-binding domain is SaCas9 or SaCas9-KKH; (xiii) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 17 and the polynucleotide-programmable nucleotide-binding domain is Nme2Cas9 -ABE8e or NmeCas9-ABE8e; (xiv) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 18 and the polynucleotide- programmable nucleotide-binding domain is Nme2Cas9 -ABE8e or NmeCas9-ABE8e; (xv) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 19 and the polynucleotide-programmable nucleotide-binding domain is SauriABE8e; (xvi) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 20 and the polynucleotide-programmable nucleotide-binding domain is SauriABE8e; (xvii) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 244 and the polynucleotide-programmable nucleotide-binding domain is Nme2Cas9 -ABE8e or NmeCas9-ABE8e; and (xviii) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 245 and the polynucleotide-programmable nucleotide-binding domain is Nme2Cas9 -ABE8e or NmeCas9-ABE8e.

[0024] In some embodiments of the system, the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 8 and the polynucleotide-programmable nucleotide- binding domain is selected from BE4max, AncBE4max, ABE8e, and ABEmax.

[0025] In some embodiments of the system, the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 3 and the polynucleotide-programmable nucleotide- binding domain is SpCas9.

[0026] In some embodiments of the system, the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 2 and the polynucleotide-programmable nucleotide- binding domain is selected from: ABEmax- VRQR, NG-ABEmax, ABEmax, xABEmax, xCas9(3.6)-ABE, and xCas9(3.7)-ABE.

[0027] In some embodiments of the system, the gRNA and the polynucleotide-programmable nucleotide-binding domain are in a ribonucleoprotein complex (RNP).

[0028] In some embodiments of the system, the gRNA is a PEgRNA and the polynucleotide- programmable nucleotide-binding domain is a prime editor polypeptide.

[0029] In some embodiments of the system, the prime editor polypeptide comprises a nickase and a reverse transcriptase.

[0030] In one aspect, this disclosure features a recombinant adeno-associated virus (rAAV) comprising: a capsid protein having at least 80% sequence identity to a sequence selected from SEQ ID NO: 212-222, 252-255; and an AAV genome comprising: a polynucleotide sequence encoding a gRNA selected from any of the gRNAs described herein; and a polynucleotide sequence encoding a polynucleotide-programmable nucleotide-binding domain.

[0031] In one aspect, this disclosure features a lipid nanoparticle (LNP) comprising; a polynucleotide sequence encoding a gRNA selected from any of the gRNAs described herein; and a polynucleotide sequence encoding a polynucleotide-programmable nucleotide-binding domain.

[0032] In one aspect, this disclosure features a lipid nanoparticle (LNP) comprising; a ribonucleoprotein complex (RNP) comprising a gRNA selected from any of the gRNAs of described herein and a polynucleotide-programmable nucleotide-binding domain.

[0033] In some embodiments of the rAAV or LNP, the polynucleotide-programmable nucleotide-binding domain is selected from; a base editor, a prime editor, a Cas9, a Cas9 nickase, a dead Cas9, a Cast 2a, a Cast 2a nickase, a dead Cast 2a, a FokI nuclease, an integrase, a zinc finger nuclease, or a transcription activator-like effector nuclease.

[0034] In some embodiments of the rAAV or LNP, the polynucleotide-programmable nucleotide-binding domain is a base editor.

[0035] In some embodiments of the rAAV or LNP, the base editor is a cytosine base editor (CBEs).

[0036] In some embodiments of the rAAV or LNP, the base editor is a adenine base editor (ABEs).

[0037] In some embodiments of the rAAV or LNP, the base editor is a non-canonical CBE base editor or a non-canonical ABE base editor.

[0038] In some embodiments of the rAAV or LNP, the polynucleotide-programmable nucleotide-binding domain is Streptococcus pyogenes Cas 9 (SpCas9) or Staphylococcus aureus (SaCas9).

[0039] In one aspect, this disclosure features an engineered cell with increased PKD1 mRNA levels, comprising: a genetic modification in the 3’ UTR of PKD1, wherein the engineered cell has increased PKD1 mRNA levels as compared to a cell that does not have a genetic modification in the 3’ UTR of PKD1.

[0040] In one aspect, this disclosure features a method for increasing PKD1 mRNA levels in a cell, the method comprising: introducing into the cell any of the systems described herein, any of the rAAV described herein, any of the LNP described herein, or a combination thereof, whereby, upon introducing the system into a cell, the system modifies a binding site of a miRNA-17 family miRNA in the 3’ UTR of PKD1, thereby preventing binding of the miRNA-17 family miRNA and de-repressing PKD1 mRNA levels.

[0041] In some embodiments, the cell is a kidney cell.

[0042] In some embodiments, the cell or kidney cell is in vivo.

[0043] In one aspect, this disclosure features a method of treating a subject having or is suspected of having a PKD-associated disorder, the method comprising: introducing into the cell any of the systems described herein, any of the rAAV described herein, any of the LNP described herein, or a combination thereof, whereby, upon introducing the system into the subject, the system modifies a binding site of a miRNA-17 family miRNA in the 3’ UTR of PKD1, thereby preventing binding of the miRNA-17 family miRNA and de-repressing PKD1 mRNA levels.

[0044] In some embodiments, the PKD-associated disorder is autosomal dominant polycystic kidney disease (ADPKD).1. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:

[0046] FIG. 1 shows an illustration of the electroporation transfection summary. FIG. 1A depicts the electroporation neon transfections carried out according to the workflow. FIG. IB depicts the FACS data plots for the representative ABE8e + gRNA8 replicate. FIG. 1C shows the sanger sequencing editing data for representative ABE8e + gRNA8 replicate. FIG. ID shows the editing efficiency in double positive cell fraction averaged from the replicate experiments, through an examination of the percent editing at the most efficiently modified base in the target window (n=3).

[0047] FIG. 2 shows a comparison of the percent editing at the first base in the target region for NGS and sanger sequencing for the ABE8e +gRNA8 DP2 neon transfection, FACS sorted and for ABE8e +gRNA8 lipofectamine transfection, all cells unsorted.

[0048] FIG. 3 depicts initial base editor screens. FIG. 3A shows the experimental workflow. FIG. B shows the fluorescent microscopy images of the lipofectamine transfected cells with ABE8e + gRNA8 to demonstrate efficiency from 6-well transfection layouts. FIG. 3C and FIG. 3D shows the heat maps of editing efficiency seen with ABEs and CBEs, respectively (ABEs n=4 (ABE8e-CP1028 &ABE8e-CP1041 : n=4 for gRNA8 and n=2 for all other gRNAs) ABE7.10 only tested with gRNA8, poor sequencing data for gRNA2 + ABE8e-CP1041). FIG. 3E describes the BEAT % editing analysis for representative replicates of the two lead ABE candidates with the target region indicated.

[0049] FIG. 4 shows next generation and single-nucleotide PAM ABEs FIG. 4A shows the next generation ABE8 percent editing in comparison to initial lead candidates (n=3). FIG. 4B shows the single-nucleotide PAM new ABEs percent editing in comparison to lead candidates (n=3).

[0050] FIG. 5 shows the next generation and single-nucleotide PAM CBEs. FIG. 5A depicts the next generation CBE6 percent editing in comparison to initial lead candidates (n=3). FIG. 5B shows the single-nucleotide PAM CBE percent editing in comparison to initial lead candidates (n=3). Note: gRNAl only has one available base in the target region.

[0051] FIG. 6 summarizes the editing at the target site by highlighting the editing efficiency achieved at each base in the target region by the lead candidate. The values are normalized to the percent editing by ABE8e + gRNA8 in the corresponding transfection. Three lead candidates are written for each base, with the lead candidate data displayed in the graph above. The initial lead candidate’s editing at both the first and second A on the graph to the right.

[0052] FIG. 7 depicts the tissue analysis for in vivo AAV delivery. FIG. 7 A shows the experimental workflow. FIG. 7B shows representative fluorescence and brightfield microscopy images of liver and kidney sections for each experimental AAV and saline negative control.AAV(Sl) denotes single-stranded AAV and scAAV(Sl) denotes self-complementary AAV. All images are 10X magnification.

[0053] FIG. 8 shows higher magnification microscopy data for single stranded AAV delivered in vivo to mice. FIG. 8A shows representative fluorescence and brightfield microscopy images of kidney sections at 40X magnification for the two strongest AAV candidates, (AAV(Sl) an AAV(S2) in comparison to AAV9 and the negative control PBS. FIG. 8B shows a quantification of the percent positive kidney tubules at 40X magnification based on counts averaged from 10 random images of kidney tissue sections.

[0054] FIG. 9 shows a schematic of a subsequence within the 3’ UTR of human PKD1 (SEQ ID NO: 11), including a binding site for miR-17 as well as the location of spacer sequences for gRNAs 1-9 (SEQ ID NO: 1-9), and gRNAs 10-21 (SEQ ID NO: 12-20, 244, 245) used to guide a polynucleotide-programmable nucleotide-binding domain to the 3’ UTR of PKD1 (SEQ ID NO: H).

[0055] FIG. 10 shows an illustration of the mechanism by which PKD1 is de-repressed in a cell following introduction of a system comprising: a gRNA capable of associating with a polynucleotide-programmable nucleotide-binding domain and guiding the polynucleotide-programmable nucleotide-binding domain to a genomic site in the 3’ UTR of PKD1; and a programmable nucleotide-binding domain.

[0056] FIG. 11 shows the results of a luciferase assay and guide RNA screen to edit each of the bases in the miR-17 binding region within the PKD1 3’ UTR (SEQ ID NO: 11). FIG. 11A-B show the results of a luciferase binding assay at 1 sec and 3 sec exposure times using plasmid expressing luciferase upstream of the PKD1 3’ UTR with various modifications to the miR-17 binding region within the 3’ UTR. The wildtype sequence of this miR-17 binding region is GCACTTTA. Asterisks denote statistical significance. mLuc = modified luciferase expression, SEAP = secreted alkaline phosphatase control. FIG. 11C-D show the editing efficiency via EditR and NGS respectively for the highest efficiency base editors for each mutation of the miR- 17 binding region. EditR is an online tool available for inferring NGS results based on Sanger sequencing data (Kluesner et al. 2018, CRISPR J). FIG. 11D-F show the ratio of expression between miR-17 treated and scrambled RNA in the luciferase assay at 1 sec and 3 sec exposure times. Ratios below 1 show that these miR-17 binding mutants are not as effective at preventing PKD1 -mediated depression. Statistical significance is shown with asterisks using standard t-test.

[0057] FIG. 12 shows the editing efficiency in three different human kidney epithelial cells with a dual AAV split-intein system for base editing. WT 9-7 and WT 9-12 cells are human kidney epithelial cells isolated from a patient with polycystic kidney disease. Results shown are for the two adenine base pairs within the GCACTTTA miR-17 binding region in the PKD1 3’ UTR.

[0058] FIG. 13 shows the fold change in PKD1 mRNA expression following treatment with a dual AAV split-intein system for base editing in human kidney epithelial cells in vitro. Data are normalized to either ACTB or GAPDH. Statistical significance between groups is shown with asterisks using standard t-test. Data are from WT 9-7 human kidney epithelial cells.

[0059] FIG. 14 shows the editing efficiency for gRNA20 (SEQ ID NO: 19) and gRNA21 (SEQ ID NO: 20) with the SauriABESe base editor compared to gRNA8 (SEQ ID NO: 8) with the ABE8e base editor in unsorted human kidney epithelial cells transfected with lipid-based transfection (Lipofectamine 3000: Thermo Fisher Scientific). Results shown are for the two adenine base pairs within the GCACTTTA miR-17 binding region in the PKD1 3’ UTR.

[0060] FIG. 15 shows the fold change in PKD1 protein expression via Western Blot in human kidney epithelial cells treated with SpCas9 + gRNA3 or SpCas9 + gRNA8 using ribonucleoprotein delivery in Fig.lSA. SpCas9 was delivered in protein form and complexed with a chemically modified guide RNA prior to delivery via electroporation. Ponceau S is used to normalize the amount of protein loaded per well. The percentages with asterisks indicate the percentage of cells in each sample with a disrupted miR-17 binding site in the PKD1 3’ UTR as indicated from next-generation sequencing in Fig. 15B.

[0061] FIG. 16 shows indel efficiency for gRNA13 (SEQ ID NO: 10), gRNA14 (SEQ ID NO: 15), gRNA15 (SEQ ID NO: 16) with SaCas9 and SaCas9-KKH delivered to human kidney epithelial cells via ribonucleoprotein delivery and lipid-based transfection respectively. SpCas9 + gRNA8 (SEQ ID NO: 8) was used as a positive control. SaCas9 and SpCas9 were delivered in protein form and complexed with a chemically modified guide RNA prior to delivery via electroporation. SaCas9-KKH was delivered with lipid-based transfection (Lipofectamine 3000: Thermo Fisher Scientific) using the MSP1830 plasmid (Addgene: 70708) and guide RNA was cloned into the BPK2660 plasmid (Addgene: 70709) and delivered.

[0062] FIG. 17 shows a time-course of indel efficiency for gRNA3 and gRNA8 following ribonucleoprotein delivery with SpCas9 in human kidney epithelial cells. Data are from WT 9-7 human kidney epithelial cells. The passage number of the cells at each timepoint is indicated in parenthesis with a p (i.e. pl, p7, etc.).2. DETAILED DESCRIPTION2.1.1. Definitions

[0063] As used herein, “base-editors” refer to a CRISPR-Cas-based genome editing technology that allows the introduction of point mutations in the DNA without generating DSBs. Two major classes of base editors have been developed: cytidine base editors or CBEs allowing OT conversions and adenine base editors or ABEs allowing A>G conversions.

[0064] The term “cargo” as used herein includes but is not limited to the group consisting of therapeutic agents, diagnostic probes, peptides, nucleic acids, antisense oligonucleotides, plasmids, proteins, nanoparticles, liposomes, chromophores, small molecules and radioactivematerials. In aspects of the invention, the cargo may also comprise any component of the CRISPR Cas system or the entire functional CRISPR Cas system. Aspects of the present invention further provide methods for delivering a desired cargo into a subject comprising: (a) preparing a complex comprising the cell -penetrating peptide of the present invention and a desired cargo, and (b) orally, intraarticularly, intraperitoneally, intrathecally, intrarterially, intranasally, intraparenchymally, subcutaneously, intramuscularly, intravenously, dermally, intrarectally, or topically administering the complex to a subject. The cargo is associated with the peptides either through chemical linkage via covalent bonds or through non-covalent interactions.

[0065] As used herein, the term "domain" or "protein domain" refers to a part of a protein sequence that may exist and function independently of the rest of the protein chain. As described in aspects of the invention, sequence identity is related to sequence homology. Homology comparisons may be conducted by eye, or more usually, with the aid of readily-available sequence comparison programs. These commercially available computer programs may calculate percent (%) homology between two or more sequences and may also calculate the sequence identity shared by two or more amino acid or nucleic acid sequences.

[0066] The term “effective amount” or “therapeutically effective amount” refers to the amount of an agent that is sufficient to effect beneficial or desired results. The therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will provide an image for detection by any one of the imaging methods described herein. The specific dose may vary depending on one or more of: the particular agent chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to be imaged, and the physical delivery system in which it is earned.

[0067] As used herein, “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, orproteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0068] As used herein, the term “guide RNA” or “gRNA” refers to an RNA sequence capable of associating with a polynucleotide-programmable nucleotide-binding domain and guiding the polynucleotide-programmable nucleotide-binding domain to a target polynucleotide sequence.

[0069] As used herein, the term “isolated” or “purified,” when used in reference to a polynucleotide, oligonucleotide, or polypeptide, means that the material is in a form other than that in which it normally is found in nature. Thus, where a polynucleotide or polypeptide occurs in a cell in nature, an isolated polynucleotide or purified polypeptide can be one that separated, at least in part, from the materials with which it is normally associated. In general, an isolated polynucleotide or a purified polypeptide is present in a form in which it constitutes at least about 5 to 10% of a composition, usually 20% to 50% of a composition, particularly about 50% to 75% of a composition, and preferably about 90% to 95% or more of a composition. Methods for isolating a polynucleotide or polypeptide are well known and routine in the art.

[0070] As used herein, the term “label” can be used to obtain a detectable (preferably quantifiable) signal and can be any atom or molecule that can bind to a nucleic acid or protein. Means a molecule. The label may provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetric analysis, X-ray diffraction or absorption, magnetism, enzyme activity, and the like. The label can be a charged moiety (positive or negative charge) or the charge can be neutral.

[0071] As used herein, the term “modification,” with reference to a nucleic acid sequence, refers to a nucleic acid sequence that comprises at least one substitution, alteration, inversion, addition, or deletion of nucleotide compared to a reference nucleic acid sequence. As used herein, the term “modification,” with reference to an amino acid sequence refers to an amino acid sequence that comprises at least one substitution, alteration, inversion, addition, or deletion of an amino acid residue compared to a reference nucleic acid sequence.

[0072] The terms “non-naturally occurring” or “engineered” are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.

[0073] As used herein, the term “nucleotide-binding domain or region” refers to the portion of a polypeptide or composition provided herein that provides specific nucleic acid binding capability. The nucleotide-binding region functions to target a subject polypeptide to specific genes.

[0074] “Prime editor” as used herein, describes a protein that is used in prime editing.

[0075] “Prime editor system” as used herein, describes the components used in prime editing.

[0076] As used herein, the term “polynucleotide” or “oligonucleotide” or “nucleotide sequence” or the like refers to a polymer of two or more nucleotides or nucleotide analogs. The polynucleotide can be a ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecule, and can be single stranded or double stranded DNA or RNA, or a double stranded DNA:RNA hybrid. A polynucleotide or oligonucleotide can contain one or more modified bases, for example, inosine or a tritylated base. The bonds linking the nucleotides in a polymer generally are phosphodiester bonds, but can be other bonds routinely used to link nucleotides including, for example, phosphorothioate bonds, thioester bonds, and the like. A polynucleotide also can be a chemically, enzymatically or metabolically modified form.

[0077] The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non amino acids. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein the term “amino acid” includes natural and / or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.

[0078] As used herein, the term “protospacer adjacent sequence” or “protospacer adjacent motif’ or “PAM” refers to an approximately 2-6 base pair DNA sequence (or a 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-long nucleotide sequence) that is an important targeting component of a Cas9 nuclease. Typically, the PAM sequence is on either strand, and is downstream in the 5' to 3' direction of Cas9 cut site. The canonical PAM sequence (i.e., the PAM sequence that is associated with the Cas9 nuclease of Streptococcus pyogenes or SpCas9) is 5'-NGG-3' wherein “N” is any nucleobase followed by two guanine (“G”) nucleobases. Different PAM sequences can be associated with different Cas9 nucleases or equivalent proteins from different organisms. In addition, any given Cas9 nuclease may be modified to alter the PAM specificity of the nuclease such that the nuclease recognizes alternative PAM sequence.

[0079] As used herein, the term “recombinant” refers to a biomolecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the gene is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, and / or (4) does not occur in nature. The term “recombinant” can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems, as well as proteins and / or mRNAs encoded by such nucleic acids. As used herein, an endogenous nucleic acid sequence in the genome of an organism (or the encoded protein product of that sequence) is deemed “recombinant” herein if a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered. In this context, a heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, whether or not the heterologous sequence is itself endogenous (originating from the same host cell or progeny thereof) or exogenous (originating from a different host cell or progeny thereof). By way of example, a promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of a host cell, such that this gene has an altered expression pattern. This gene would now become “recombinant” because it is separated from at least some of the sequences that naturally flank it. A nucleic acid is also considered “recombinant” if it contains any modifications that do not naturally occur to the corresponding nucleic acid in a genome. For instance, an endogenous coding sequence is considered “recombinant” if it contains an insertion, deletion or a point mutation introduced artificially, e.g.,by human intervention. A “recombinant nucleic acid” also includes a nucleic acid integrated into a host cell chromosome at a heterologous site and a nucleic acid construct present as an episome.

[0080] As used herein, the term “region” as used herein, refers to a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein.

[0081] As used herein, the term “synthetic" is used to refer to an entity that is made is lab- created and not naturally produced or isolated, without modification, from a naturally occurring source. A recombinant polymer, such as a recombinant polynucleotide or polypeptide, may be synthetic. Synthetic polymers such as polynucleotides or polypeptides may be produced by any method known to those of skill in the art, including but not limited to solid phase synthesis, solution phase synthesis, biological synthesis by, e.g., host cells, etc.

[0082] As used herein, the term “subject” is a mammal. A subject may be a human or nonhuman mammal. Given context, a subject may be used interchangeably with patient, individual, donor, etc.

[0083] As used herein, the term “target”, “target region", or “target site” refers to a protein or functional portion or variant thereof. A target may be expressed on the surface of a particular cell (a “target cell”) or expressed within (e.g., on the surfaces of) cells in a population of cells. A target may have a certain percent identity to a reference protein and still be referred to as a target by a particular name (e.g., miR17 binding region). A target may be or comprise a binding region, such as an epitope or target region, to which a polynucleotide (e.g., a polynucleotide- programmable nucleotide-binding domain) of the present disclosure binds.

[0084] The terms “therapeutic agent”, “therapeutic capable agent” or “treatment agent" are used interchangeably and refer to a molecule or compound that confers some beneficial effect upon administration to a subject. The beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.

[0085] As used herein, the term “treatment” (as well as “treat” or “treating”) refers to partial or complete alleviation, amelioration, mitigation, prevention, reduction in risk of onset, relief, inhibition, delay in onset of, reduction in severity of, reduction in frequency or incidence of one or more causes, features, and / or symptoms of or associated with a particular disease, disorder, and / or condition.

[0086] As used herein the term “variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature.

[0087] As used herein the term “wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.2.2. Guide RNAs

[0088] This disclosure features guide RNAs for targeting the 3’ UTR of the polycystin 1(PKD1). In the present disclosure, the gRNA is capable of associating with a polynucleotideprogrammable nucleotide-binding domain and guiding the polynucleotide-programmable nucleotide-binding domain to a target polynucleotide sequence. In this case, the target polynucleotide sequence is a genomic site in the 3’ UTR of PKD1 (SEQ ID NO: 11).

[0089] PKD1 3' UTR (Human) has a sequence as shown below.

[0090] Twenty-one guide RNAs that target the miR-17 human sequence were identified (see Table 1). These guide RNA sequences were selected based on having a C->T or A->G edit in positions 4-9 starting from the 5’ end of a guide RNA placed immediately upstream of a PAM (NG, NNGRRT, or TTTV). For example, Position 3 corresponds to the third nucleotide in the guide RNA reading from the 5’ direction.

[0091] In one embodiment, a guide RNA (gRNA) includes a spacer sequence complementary to a genomic site in 3’ UTR of PKD1; and a scaffold capable of associating with a polynucleotide- programmable nucleotide-binding domain and guiding the polynucleotide-programmable nucleotide-binding domain to the genomic site in the 3’ UTR of PKD1. A list of non-limiting scaffolds can be seen as described in Table Z.

[0092] In another embodiments, a guide RNA (gRNA) includes: a spacer sequence complementary to at least 17, 18, 19, or 20 contiguous nucleotides of the sequence of SEQ ID NO: 11 ; and a scaffold capable of associating with a polynucleotide-programmable nucleotide- binding domain and guiding the polynucleotide-programmable nucleotide-binding domain to a sequence complementary to the spacer sequence. In some embodiments, the 3 ’UTR PKD1 includes a sequence of SEQ ID NO: 11, and the gRNA is designed to have a spacer sequence that enables the gRNA once associated with a polynucleotide-programmable nucleotide-binding domain to guide the polynucleotide-programmable nucleotide-binding domain to the 3’ UTR of PKD1. In some embodiments, the guide RNA (gRNA) includes: a spacer sequencecomplementary to at least 17 contiguous nucleotides of the sequence of SEQ ID NO: 11. In some embodiments, the guide RNA (gRNA) includes: a spacer sequence complementary to at least 18 contiguous nucleotides of the sequence of SEQ ID NO: 11. In some embodiments, the guide RNA (gRNA) includes: a spacer sequence complementary to at least 18 contiguous nucleotides of the sequence of SEQ ID NO: 11. In some embodiments, the guide RNA (gRNA) includes: a spacer sequence complementary to at least 19 contiguous nucleotides of the sequence of SEQ ID NO: 11. In some embodiments, the guide RNA (gRNA) includes: a spacer sequence complementary to at least 20 contiguous nucleotides of the sequence of SEQ ID NO: 11.

[0093] In one embodiment, a guide RNA (gRNA) includes: a spacer sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 1-9 and SEQ ID NO: 12-20, 244, 245; and a scaffold capable of associating with a polynucleotide-programmable nucleotide- binding domain and guiding the polynucleotide-programmable nucleotide-binding domain to a sequence complementary to the spacer sequence.

[0094] In some embodiments, the gRNA includes one or more modified nucleotides. In some embodiments, the one or more modifications increase stability of the gRNA, for example, when the gRNA is introduced into a cell.

[0095] In some embodiments, the gRNA is a synthetic gRNA.

[0096] In some embodiments, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NOs: 1-10, 12-20, 244, 245 (see Table 1).

[0097] In some embodiments, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 3. In some embodiments, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 4. In some embodiments, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 5. In some embodiments, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 6. In some embodiments, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 7. In some embodiments, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 8. In some embodiments, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 9. In some embodiments, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 10. In some embodiments, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 12. In some embodiments, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 13. In some embodiments, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 14. In some embodiments, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 15. In some embodiments, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 16. In some embodiments, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 17. In some embodiments, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 18. In some embodiments, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 19. In some embodiments, thespacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 20. In some embodiments, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 244. In some embodiments, the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 245.

[0098] In some embodiments, the spacer comprises a sequence of SEQ ID NO: 1. In some embodiments, the spacer comprises a sequence of SEQ ID NO: 2. In some embodiments, the spacer comprises a sequence of SEQ ID NO: 3. In some embodiments, the spacer comprises a sequence of SEQ ID NO: 4. In some embodiments, the spacer comprises a sequence of SEQ ID NO: 5. In some embodiments, the spacer comprises a sequence of SEQ ID NO: 6. In some embodiments, the spacer comprises a sequence of SEQ ID NO: 7. In some embodiments, the spacer comprises a sequence of SEQ ID NO: 8. In some embodiments, the spacer comprises a sequence of SEQ ID NO: 9. In some embodiments, the spacer comprises a sequence of SEQ ID NO: 10. In some embodiments, the spacer comprises a sequence of SEQ ID NO: 12. In some embodiments, the spacer comprises a sequence of SEQ ID NO: 13. In some embodiments, the spacer comprises a sequence of SEQ ID NO: 14. In some embodiments, the spacer comprises a sequence of SEQ ID NO: 15. In some embodiments, the spacer comprises a sequence of SEQ ID NO: 16. In some embodiments, the spacer comprises a sequence of SEQ ID NO: 17. In some embodiments, the spacer comprises a sequence of SEQ ID NO: 18. In some embodiments, the spacer comprises a sequence of SEQ ID NO: 19. In some embodiments, the spacer comprises a sequence of SEQ ID NO: 20. In some embodiments, the spacer comprises a sequence of SEQ ID NO: 244. In some embodiments, the spacer comprises a sequence of SEQ ID NO: 245.2.2.1. Prime Editing Guide RNA (PEgRNA)

[0099] In some embodiments, the gRNA also includes the components of a prime editing guide RNA (pegRNA). For example, a PEgRNA comprises one or more of a primer binding site, and a reverse transcriptase template that includes one or more nucleotide changes compared to the polynucleotide sequence at the genomic site. The primer binding site includes a region of complementarity to a region downstream of a nick site at the genomic site, where the nick is made by the primer editor protein (e.g., a Cas9 fused to a reverse transcriptase).

[0100] Non-limiting example of PEgRNAs and PEgRNA structure are as described in U.S. Pat. Nos. 11,643,652 and U.S. Patent Publication No. US 2022 / 0090064A1, each of which is hereby incorporated by reference in its entirety. In one non-limiting example, Anzalone et al., 2019 (Nature 576: 149) describes prime editing and a prime editing complex using a type II CRISPR and can be used herein. A prime editing complex consists of a type II CRISPR PE protein containing an RNA-guided DNA-nicking domain fused to a reverse transcriptase (RT) domain and complexed with a pegRNA. The pegRNA comprises (5’ to 3’) a spacer that is complementary to the target sequence of a genomic DNA, a nickase (e.g. Cas9) binding site, a reverse transcriptase template including editing positions (edits to be introduced into the genomic site), and a primer binding site (PBS). The PE-pegRNA complex binds the target DNA and the CRISPR protein nicks the PAM-containing strand. The resulting 3’ end of the nicked target hybridizes to the primer-binding site (PBS) of the pegRNA, then primes reverse transcription of new DNA containing the desired edit using the RT template of the pegRNA. The overall structure of the pegRNA is like that of a typical type II sgRNA with a reverse transcriptase template / primer binding site appended to the 3’ end. The structure leaves the PBS at the 3’ end of the pegRNA free to bind to the nicked strand complementary to the target which forms the primer for reverse transcription.

[0101] Guide RNAs of CRISPRs differ in overall structure. For example, while the spacer of a type II gRNA is located at the 5’ end, the spacer of a type V gRNA is located towards the 3’ end, with the CRISPR protein (e.g. Casl2a) binding region located toward the 5’ end. Accordingly, the regions of a type V pegRNA are rearranged compared to a type II pegRNA. The overall structure of the pegRNA is like that of a typical type II sgRNA with a reverse transcriptase template / primer binding site appended to the 3’ end. The pegRNA comprises (5’ to 3’) a CRISPR protein-binding region, a spacer which is complementary to the target sequence of a genomic DNA, a reverse transcriptase template including editing positions, and primer binding site (PBS).2.3. Systems

[0102] This disclosure also features systems for inhibiting a miRNA-17 family miRNA (e.g., miR-17) from binding to the 3’ UTR of PKD1.

[0103] In some embodiments, a system for inhibiting a miRNA-17 family miRNA (e.g., miR-17) from binding to the 3’ UTR of PKD1 includes: a gRNA (e.g., any of the gRNA described in Section 4.1) capable of associating with a polynucleotide-programmable nucleotide-binding domain and guiding the polynucleotide-programmable nucleotide-binding domain to a genomic site in the 3’ UTR of PKD1; and a programmable nucleotide-binding domain or a polynucleotide encoding the polynucleotide programmable nucleotide-binding domain.

[0104] In some embodiments, a system for inhibiting a miRNA-17 family miRNA (e.g., miR-17) from binding to the 3’ UTR of PKD1 includes: a gRNA capable of associating with a polynucleotide-programmable nucleotide-binding domain and guiding the polynucleotide- programmable nucleotide-binding domain to a genomic site in SEQ ID NO: 11; and a programmable nucleotide-binding domain or a polynucleotide encoding the polynucleotide programmable nucleotide-binding domain.

[0105] In some embodiments, the system including gRNAs capable of associating with a polynucleotide-programmable nucleotide-binding domain and guiding the polynucleotide- programmable nucleotide-binding domain to a genomic site in SEQ ID NO: 11, includes variants thereof including 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater to SEQ ID NO: 11.

[0106] In some embodiments, a system for inhibiting a miRNA-17 family miRNA (e.g., miR-17) from binding to the 3’ UTR of PKD1 includes: a gRNA having a spacer sequence comprising a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 1-10, 12-20, 244, 245; and a programmable nucleotide-binding domain or a polynucleotide encoding the polynucleotide programmable nucleotide-binding domain.

[0107] In some embodiments, the systems described herein are used to either directly or indirectly inhibit a miRNA-17 family miRNA from binding to the 3’ UTR of PKD1. For example, in some embodiments, upon introducing the system into a cell, the system genetically modifies a binding site of a miRNA-17 family miRNA in the 3’ UTR of PKD1, thereby preventing binding of the miRNA-17 family miRNA and de-repressing PKD1 mRNA.2.3.1. Polynucleotide-Programmable Nucleotide-Binding Domain (PPNBD)

[0108] In some embodiments, wherein the polynucleotide-programmable nucleotide-binding domain is selected from: a base editor, a prime editor, a Cas9, a Cas9 nickase, a dead Cas9, a Cast 2a, a Casl2a nickase, a dead Casl2a, a FokI nuclease, an integrase, a zinc finger nuclease, or a transcription activator-like effector nuclease.2.3.1.1 Base Editors

[0109] In some embodiments, the polynucleotide-programmable nucleotide-binding domain is a base editor. Base editing is a promising therapeutic strategy for genetic diseases caused by point mutations and might be more effective than approaches based on homology-directed repair or knockout based approaches. In some embodiments, one or more base editors are selected from Table 2.

[0110] In some embodiments, the base editor is a cytosine base editor (CBEs).

[0111] In some embodiments, the base editor is a adenine base editor (ABEs).

[0112] In some embodiments, the base editor is a non-canonical CBE base editor or a non- canonical ABE base editor.

[0113] In some embodiments, a base editor is selected from; ABEmax-VRQR, ABEmax-NG, ABEmax, xABEmax, xCas9(3.6)-ABE, xCas9(3.7)-ABE, CP-CBEmax, CP-ABEmax, BE3- xCas9-3.6, BE3-xCas9-3.7, BE4max-xCas9(3.6), BE4max-xCas9(3.7)-, BE4max, BE4max- CP1028, BE3-pBK-VQR, AncBE4max, CBE6a, CBE6b, CBE6c, CBE6d, ABE8e, ABE8e-NG, ABE8e-CP1028, pCMV-SpRY-ABE8e, ABE8.20-d, ABE8.20-m, ABE8.17-m, ABE8e-CP1041, ABEmax-CP1012, ABE7.10-3.6, ABE7.10-3.7, ABE7.10, and pCAG-CBE4max-SpRY.

[0114] In some embodiments, a base editor used herein is as described in Koblan et al. (Nat. Bio. volume 36, pages843-846 (2018)), which is hereby incorporated by reference in its entirety. For example, Koblan teaches ABEmax, BE4max, and AncBE4max editors, including variants thereof.

[0115] In some embodiments, a base editor used herein is as described in (Kim et al., Nat.Biotech. 35, 371-376 (2017)), which is hereby incorporated by reference in its entirety. Theteachings of Kim et al. increasing the genome-targeting scope of base editing. For example, Kim teaches CBEs with new PAM types (e.g., NGA, NGAG, NGCG, NNNRRT) and CBEs with shortened editing windows to reduce bystander edits.

[0116] In some embodiments, a base editor used herein is as described in Huang et al., (Nat. Biotech. 37, 626-631 (2019)), which is hereby incorporated by reference in its entirety.Circularly permuted and PAM-modified Cas9 variants. Huang teaches ABEs with new PAM types (NG, NGA, NGCG, NNNRRT). Huang also teaches CP-ABEmax and CP-CBEmax editors with expanded editing windows.

[0117] In some embodiments, a base editor used herein is as described in Richter et al., (Nat.Biotech. 38, 883-891 (2020)), which is hereby incorporated by reference in its entirety. Richter teaches phage-assisted evolution of an adenine base editor. For example, Richter teaches ABE8e.Richter applied this new ABE8e to non-canonical PAMs and broader editing windows (SaABE8e, LbABE8e, enAsABE8e, NG-Abe8e, SaKKH-ABE8e, and CP-ABE8e)

[0118] In some embodiments, a base editor used herein is as described in Hu et al., (Nature 556, 57-63 (2018)), which is hereby incorporated by reference in its entirety. Hu teaches evolved Cas9 variants with broad PAM compatibility and DNA specificity. For example, Hu teaches ABEs & CBEs with NG PAMs (e.g., xCas9-3.7, xCas9-3.6).

[0119] In some embodiments, a base editor used herein is as described in Yu et al., (Nat Communications 2020), which is hereby incorporated by reference in its entirety. Yu teaches cytosine base editors with minimized off-target editing. For example, Yu teaches CBEs with reduced off-target editing (NGG, NGGRRT PAMs).

[0120] In some embodiments, a base editor used herein is as described in Gaudelli et al., (Nature Biotechnology, 38:892-900 (2020)), which is hereby incorporated by reference in its entirety. Gaudelli teaches the directed evolution of adenine base editors with increased activity and therapeutic application, For example, Gaudelli teaches the further evolution of ABE7.10 to create ABE8s with higher editing efficiencies at challenging loci in primary human cells to achieve 98-99% target modification.

[0121] In some embodiments, a base editor used herein is as described in Zhang et al., (Nat Communications, 15: 1697 (2024)), which is hereby incorporated by reference in its entirety. Zhang teaches the phage-assisted evolution of highly active cytosine base editors with enhanced selectivity and minimal sequence context preference over CBEmax. For example, Zhang teaches the evolution of CBE6s such as CBE6a, CBE6b, CBE6c, and CBE6d with SpCas9, from a TadA-mediated dual cytosine and adenine base editor, showing higher selectivity and enhanced sequence-context compatibility.

[0122] In some embodiments, a base editor used herein is as described in Lam et al., (Nature Biotechnology, 41 :686-697 (2023)), which is hereby incorporated by reference in its entirety. Lam teaches improved cytosine base editors such as CBE-T and CABE-T editors derived from TadA ABE variants. For example, Lam describes CBE-T and CABE-T editors such as CBE- T1.46, CBE-T1.14, and CBE-T1.52, which are able to undergo both ABE and CBE editing simultaneously.

[0123] In some embodiments, a base editor used herein is as described in Neugebauer et al., (Nature Biotechnology, 41 :673-685 (2023)), which is hereby incorporated by reference in its entirety. Neugebauer teaches the evolution of adenine base editor into cytosine editor. Specifically, Neugebauer describes the evolution of an ABE into a CBE, such as SPCas9 TadCBEd and TadCBEd-V106W for the expansion and utility of CBEs for precision gene editing.Table 2 describes exemplary base editors.2.3.1.2 Type II CRISPR Proteins (e.g., Cas9)

[0124] In some embodiments, the polynucleotide-programmable nucleotide-binding domain is a Type II CRISPR protein. In some embodiments, the polynucleotide-programmable nucleotide- binding domain is a Streptococcus pyogenes Cas 9 (SpCas9) or a Staphylococcus aureus (SaCas9). In some embodiments, a SaCas9-based system (e.g., a SaCas9-gRNAl) is used for in vivo editing.

[0125] In some embodiments, the polynucleotide-programmable nucleotide-binding domain comprises a Streptococcus pyogenes Cas 9 (SpCas9). Streptococcus pyogenes Cas9 (SpCas9), the most common enzyme used in genome-editing applications, is a large nuclease of 1368 amino acid residues. Advantages of SpCas9 include its short, 5'-NGG-3' PAM and very high average editing efficiency. SpCas9 consists of two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe. The REC lobe can be divided into three regions, a long a helix referred to as the bridge helix (residues 60-93), the RECI (residues 94-179 and 308-713) domain, and the REC2 (residues 180-307) domain. The NUC lobe consists of the RuvC (residues 1-59, 718— 769, and 909-1098), HNH (residues 775-908), and PAM-interacting (PI) (residues 1099-1368) domains. The negatively charged sgRNA:target DNA heteroduplex is accommodated in a positively charged groove at the interface between the REC and NUC lobes. In the NUC lobe, the RuvC domain is assembled from the three split RuvC motifs (RuvC I— III) and interfaces with the PI domain to form a positively charged surface that interacts with the 30 tail of the sgRNA. The HNH domain lies between the RuvC II— III motifs and forms only a few contacts with therest of the protein. Structural aspects of SpCas9 are described by Nishimasu et al., Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA, Cell 156, 935-949 (2014).

[0126] In some embodiments, Cas9 proteins that are smaller than SpCas9 nice are preferred. Cas9 proteins smaller than SpCas9 allow more efficient packaging of nucleic acids encoding CRISPR systems, e.g., Cas9 and sgRNA into one rAAV (“all-in-one-AAV”) particle. In addition, efficient packaging of CRISPR systems can be achieved in other viral vector systems (i.e., lentiviral, integration deficient lentiviral, hd-AAV, etc.) and non-viral vector systems (i.e., lipid nanoparticle). Small Cas9 proteins can be advantageous for multidomain-Cas-nuclease- based systems for nicking, inducing indels, or prime editing.

[0127] In some embodiments, the polynucleotide-programmable nucleotide-binding domain comprises a Staphylococcus aureus Cas9 (SaCas9 or SauCas9, 1053 amino acid residues). In some embodiments, the polynucleotide-programmable nucleotide-binding domain comprises a Campylobacter jejuni Cas9 (CjCas9, 984 amino residues). Both SaCas9 and CjCas9 recognize longer PAMs, 5'-NNGRRT-3' for SauCas9 (R = A or G) and 5'-NNNNRYAC-3' for CjCas9 (Y = C or T), which reduces the number of uniquely addressable target sites in the genome, in comparison to the NGG SpCas9 PAM.

[0128] In some embodiments, the polynucleotide-programmable nucleotide-binding domain comprises a Staphylococcus lugdunensis (Siu) Cas9 (SluCas9). Overall, the small Cas9s and nickases are useful in the instant disclosure.

[0129] In some embodiments, the polynucleotide-programmable nucleotide-binding domain comprises a “Cas9 variant” having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference Cas9 protein, including any wild type Cas9, or mutant Cas9 (e.g., a dead Cas9 or Cas9 nickase), or fragment Cas9, or circular permutant Cas9, or other variant of Cas9 disclosed herein or known in the art. In some embodiments, a Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to a reference Cas9. In someembodiments, the Cas9 variant comprises a fragment of a reference Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9.

[0130] In some embodiments, the disclosure also may utilize Cas9 fragments that retain their functionality and that are fragments of any herein disclosed Cas9 protein. In some embodiments, the Cas9 fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length.

[0131] In various embodiments, the base editors or prime editors disclosed herein may comprise one of the Cas9 variants described as follows, or a Cas9 variant thereof having at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any reference Cas9 variants.2.3.1.3 Table 3 describes Cas9 orthologs.2.3.1.4 Type V CRISPR Proteins

[0132] In some embodiments, the polynucleotide-programmable nucleotide-binding domain is a Type V CRISPR protein. In some embodiments, the polynucleotide-programmable nucleotide- binding domain is a Casl2 protein.

[0133] In some embodiments, the type V CRISPR family includes Francisella novicida U112 Cpfl (FnCpfl) also known as FnCasl2a. FnCpfl adopts a bilobed architecture with the two lobes connected by the wedge (WED) domain. The N-terminal REC lobe consists of two a- helical domains (RECI and REC2) that have been shown to coordinate the crRNA-target DNA heteroduplex. The C-terminal NUC lobe consists of the C-terminal RuvC and Nuc domains involved in target cleavage, the arginine-rich bridge helix (BH), and the PAM-interacting (PI) domain. The repeat-derived segment of the crRNA forms a pseudoknot stabilized by intramolecular base-pairing and hydrogen-bonding interactions. The pseudoknot is coordinated by residues from the WED, RuvC, and REC2 domains, as well as by two hydrated magnesium cations. Notably, nucleotides 1-5 of the crRNA are ordered in the central cavity of FnCasl2a and adopt an A-form-like helical conformation. Conformational ordering of the seed sequence is facilitated by multiple interactions between the ribose and phosphate moieties of the crRNA backbone and FnCpfl residues in the WED and RECI domains. These include residues Thrl6, Lys595, His804, and His881 from the WED domain and residues Tyr47, Lys51, Phel82, and Argl86 from the RECI domain. The structure of the FnCasl2a-crRNA complex further reveals that the bases of the seed sequence are solvent exposed and poised for hybridization with target DNA. Structural aspects of FnCpfl are described by Swarts et al., Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Casl2a, Molecular Cell 66, 221-233, April 20, 2017.

[0134] In some embodiments, the type V CRISPR family is a LbCasl2a. In some embodiments, the polynucleotide-programmable nucleotide-binding domain is a LbCasl2a.

[0135] In some embodiments, the type V CRISPR is AsCpfl from Acidaminococcus sp BV3L6 (Yamano et al., Crystal structure of Cpfl in complex with guide RNA and target DNA, Cell 165, 949-962, May 5, 2016).

[0136] In certain embodiments, the nuclease comprises a Casl2f effector. Small CRISPR- associated effector proteins belonging to the type V-F subtype have been identified through the mining of sequence databases and members classified into Casl2fl (Casl4a and type V-U3), Casl2f2 (Casl4b) and Casl2f3 (Casl4c, type V-U2 and U4). (See, e.g., Karvelis et al., PAM recognition by miniature CRISPR-Casl2f nucleases triggers programmable double-strandedDNA target cleavage. Nucleic Acids Research, 21 May 2020, 48(9), 5016-23 doi.org / 10.1093 / nar / gkaa208). Xu et al. described development of a 529 amino acid Casl2f- based system for mammalian genome engineering through multiple rounds of iterative protein engineering and screening. (Xu, X. et al., Engineered Miniature CRISPR-Cas System for Mammalian Genome Regulation and Editing. Molecular Cell, October 21, 2021, 81(20): 4333- 45, doi.org / 10.1016 / j.molcel.2021.08.008).

[0137] Exemplary CRISPR-Cas proteins and enzymes used herein include the following without limitation.2.3.1.5 Protospacer Adjacent Motif

[0138] With reference to the canonical SpCas9 amino acid sequence, for example, the PAM specificity can be modified by introducing one or more mutations, including (a) DI 135V, R1335Q, and T1337R “the VQR variant”, which alters the PAM specificity to NGAN or NGNG, (b) DI 135E, R1335Q, and T1337R “the EQR variant”, which alters the PAM specificity to NGAG, and (c) DI 135V, G1218R, R1335E, and T1337R “the VRER variant”, which alters the PAM specificity to NGCG. In addition, the DI 135E variant of canonical SpCas9 still recognizes NGG, but it is more selective compared to the wild type SpCas9 protein.

[0139] It will also be appreciated that Cas9 enzymes from different bacterial species (i.e., Cas9 orthologs) can have varying PAM specificities and in some embodiments are therefore chosen based on the desired PAM recognition. For example, Cas9 from Staphylococcus aureus (SaCas9) recognizes NGRRT or NGRRN. In addition, Cas9 from Neisseria meningitis (NmCas) recognizes NNNNGATT. In another example, Cas9 from Streptococcus thermophilis (StCas9) recognizes NNAGAAW. In still another example, Cas9 from Treponema denticola (TdCas) recognizes NAAAAC. These examples are not meant to be limiting. It will be further appreciated that non-SpCas9s bind a variety of PAM sequences, which makes them useful to expand the range of sequences that can be targeted according to the invention. Furthermore, non-SpCas9s may have other characteristics that make them more useful than SpCas9. For example, Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase smaller than SpCas9, so it can be packaged into adeno-associated virus (AAV).2.3.1.6 Non-Limiting System Examples

[0140] In some embodiments, the system includes combinations of gRNA and polynucleotide- programmable nucleotide-binding domain selected from:(i) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 1 and the polynucleotide-programmable nucleotide-binding domain is VQR-BE3;(ii) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 2 and the polynucleotide-programmable nucleotide-binding domain is selected from: ABEmax -VRQR, NG-ABEmax, ABEmax, xABEmax, xCas9(3.6)-ABE, and xCas9(3.7)- ABE;(iii) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 3 and the polynucleotide-programmable nucleotide-binding domain is SpCas9;(iv) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 5 and the polynucleotide-programmable nucleotide-binding domain is selected from: CP-CBEmax, NG-ABEmax, and CP-ABEmax;(v) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 7 and the polynucleotide-programmable nucleotide-binding domain is selected from: VRQR, NG, xBE, xCas9(3.6)-ABE, xCas9(3.6)-CBE, xCas9(3.7)-ABE, and xCas9(3.7)- CBE;(vi) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 8and the polynucleotide-programmable nucleotide-binding domain is selected from BE4max, AncBE4max, ABE8e, and ABEmax;(vii) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 9 and the polynucleotide-programmable nucleotide-binding domain is all non- canonical ABE / CBE editors; and(viii) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 10 and the polynucleotide-programmable nucleotide-binding domain is SaCas9 or SaCas9-KKH;(ix) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 12 and the polynucleotide-programmable nucleotide-binding domain is SpCas9;(x) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 13 and the polynucleotide-programmable nucleotide-binding domain is SpCas9;(xi) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 14 and the polynucleotide-programmable nucleotide-binding domain is SpCas9;(xii) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 15 and the polynucleotide-programmable nucleotide-binding domain is SaCas9 or SaCas9-KKH;(xiii) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 16 and the polynucleotide-programmable nucleotide-binding domain is SaCas9 or SaCas9-KKH;(xiv) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 17 and the polynucleotide-programmable nucleotide-binding domain is Nme2Cas9 -ABE8e or NmeCas9-ABE8e;(xv) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 18 and the polynucleotide-programmable nucleotide-binding domain is Nme2Cas9 -ABE8e or NmeCas9-ABE8e;(xvi) the gRNA comprises a spacer sequence having at least 80% sequence identity toSEQ ID NO: 19 and the polynucleotide-programmable nucleotide-binding domain is SauriABE8e;(xvii) the gRNA comprises a spacer sequence having at least 80% sequence identity toSEQ ID NO: 20 and the polynucleotide-programmable nucleotide-binding domain is SauriABESe;(xviii) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 244 and the polynucleotide-programmable nucleotide-binding domain is Nme2Cas9 -ABE8e or NmeCas9-ABE8e; and(xix) the gRNA comprises a spacer sequence having at least 80% sequence identity toSEQ ID NO: 245 and the polynucleotide-programmable nucleotide-binding domain isNme2Cas9 -ABE8e or NmeCas9-ABE8e.

[0141] In some embodiments, the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 8 and the polynucleotide-programmable nucleotide-binding domain is selected from BE4max, AncBE4max, ABE8e, and ABEmax.

[0142] In some embodiments, the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 3 and the polynucleotide-programmable nucleotide-binding domain is SpCas9.

[0143] In some embodiments, the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 2 and the polynucleotide-programmable nucleotide-binding domain is selected from: ABEmax-VRQR, NG-ABEmax, ABEmax, xABEmax, xCas9(3.6)- ABE, and xCas9(3.7)-ABE.

[0144] As a non-limiting example, useful adenine base editors include VRQR- ABEmax, NG- ABEmax, NG-ABE8e, xABEmax, xCas9(3.6)-ABE7.10, xCas9(3.7)-ABE7.10, ABE8e, ABEmax, CP1028-ABE8e, CP1041-ABE8e, CP1012-ABEmax, CP 1028 -ABEmax, CP1041-ABEmax, CP 1249- ABEmax, CP 1300 -ABEmax, ABE8.20-m, ABE8.17-m, ABE8.20-d,ABE8.13-m, ABE8.8-m, ABE8.13-d, ABE8.8-d, and ABE8.17-d.

[0145] In further embodiments, a non-limiting example of useful cytosine base editors include BE4max (CBEmax), AncBE4max, CP1012-CBEmax, CP1028-CBEmax, CP1041-CBEmax, CP1249-CBEmax, xCas9(3.6)-BE4, xCas9(3.6)-BE3, xCas9(3.7)-BE4, xCas9(3.7)-BE3, VQR- BE3, CBE6a, CBE6b, CBE6c, and CBE6d.

[0146] In some embodiments, the system includes a combination of gRNA and editors as described in Table 8.

[0147] In some embodiments, the gRNA and the polynucleotide-programmable nucleotide- binding domain are in a ribonucleoprotein complex (RNP).2.3.1.7 Prime Editor Systems

[0148] Also provided herein are prime editor systems for inhibiting a miRNA-17 family miRNA from binding to the 3’ UTR of PKD1, whereby the prime editor system generate a prime edit that inhibits binding of a miRNA-17 family miRNA (e.g., miR-17) binding to the 3’ UTR of PKD1.

[0149] In some embodiments, the system includes a prime editing guide RNA (PEgRNA) comprising a spacer sequence complementary to a genomic site in 3’ UTR of PKD1 (see, e.g.,SEQ ID NO: 11), a scaffold capable of associating with prime editor protein and guiding the prime editor protein to the genomic site in the 3’ UTR of PKD1 (see, e.g., any of the PEgRNA described in Section 4.1.1), and a prime editor protein, whereby the prime editor system generate a prime edit that inhibits binding of a miRNA-17 family miRNA (e.g., miR-17) binding to the 3’UTR ofPKDl.

[0150] A noncomprehensive and non-limiting list of various scaffolds is depicted in Table Z.

[0151] In some embodiments, the system comprising a prime editing system includes a PEgRNA that comprises a spacer sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 1-10, and SEQ ID NO: 12-20, 244, 245; and a prime editor protein, whereby the prime editor system generate a prime edit that inhibits binding of a miRNA-17 family miRNA (e.g., miR-17) binding to the 3’ UTR of PKD1.

[0152] Prime editing uses CRISPR enzyme that nicks or cuts only single strand of double stranded DNA, i.e., a nickase; the nickase can occur either naturally or by mutation or modification of a nuclease that makes double stranded cuts. The nickase is programmed (directed) with a prime-editing guide RNA (pegRNA). The skilled person in the art would appreciate that the pegRNA both specifies the target site and encodes the desired edit. The nickase may be programmed (directed) with an pegRNA. Advantageously the nickase is a catalytically-impaired Cas9 endonuclease, a Cas9 nickase, that is fused to the reverse transcriptase. During genetic editing, the Cas9 nickase part of the protein is guided to the DNA target site by the atgRNA (pegRNA), whereby a nick or single stranded cut occurs. The reverse transcriptase domain then uses the atgRNA (pegRNA) to template reverse transcription of the desired edit, directly polymerizing DNA onto the nicked target DNA strand. The edited DNA strand replaces the original DNA strand, creating a heteroduplex containing one edited strand and one unedited strand. Afterward, optionally, the prime editor (PE) guides resolution of the heteroduplex to favor copying the edit onto the unedited strand, completing the process (typically achieved with a nickase gRNA.

[0153] In some embodiments, inhibiting a miRNA-17 family miRNA from binding to the 3’ UTR of PKD1 includes deleting and / or replacing all or a portion of the target sequence (e.g., the genomic site. In some embodiments, the DNA deletion replacement is induced using a pair of pegRNA that target opposite DNA strands, programming not only the sites that are nicked but also the outcome of the repair (i.e., PrimeDel by Choi et al. Nat. Biotechnology, October 14,2021; Choi et al. is incorporated herein by reference and TwinPE by Anzalone et cz / .BioRxiv, November 2, 2021; Anzalone et al. is incorporated herein by reference). In some embodiments described herein, the DNA deletion is induced using a single pegRNA. In some embodiments, the DNA deletion and replacement is induced using a wild type Cas9 prime editor (PE-Cas9) system (i.e., PED AR by Jiang et al. Nat. Biotechnology, October 14, 2021; Jiang et al. is incorporated herein by reference in its entirety). In certain embodiments, the constructs and methods described herein may be utilized to incorporate the pair of pegRNAs used in PrimeDel, TwinPE (WO2021226558 incorporated by reference herein in its entirety), or PED AR, the prime editor polypeptide, and optionally a nickase guide RNA (ngRNA).

[0154] In some embodiments, the prime editors can refer to a retrovirus or lentivirus reverse transcriptase such as a Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (RT) fused to a CRISPR enzyme nickase such as a Cas9 H840A nickase, a Cas9nickase. In some embodiments the RT can be fused at, near or to the C -terminus of a Cas9nickase, e.g., Cas9 H840A. Fusing the RT to the C -terminus region, e.g., to the C -terminus, of the Cas9 nickase may result in higher editing efficiency. In some embodiments, the CRISPR enzyme nickase, e.g., Cas9(H840A), i.e., a Cas9nickase, can be linked to a non-M-MLV reverse transcriptase such as an AMV-RT or XRT (Cas9(H840A)-AMV-RT or XRT). In some embodiments, instead of the CRISPR enzyme nickase being a Cas9 (H840A), i.e., instead of being a Cas9 nickase, the CRISPR enzyme nickase instead can be a CRISPR enzyme that naturally is a nickase or cuts a single strand of double stranded DNA; for instance, the CRISPR enzyme nickase can be Casl2a / b. Alternatively, the CRISPR enzyme nickase can be another mutation of Cas9, such as Cas9(D10A). A CRISPR enzyme, such as a CRISPR enzyme nickase, such as Cas9 (wild type), Cas9(H840A), Cas9(D10A) or Cas 12a / b nickase can be fused in some embodiments to a pentamutant of M-MLV RT (D200N / L603W / T33OP / T306K / W313F), whereby there can be up to about 45-fold higher efficiency, and this is called PE2. In some embodiments, the M-MLV RT comprise one or more of the mutations Y8H, P51L, S56A, S67R, E69K, V129P, L139P, T197A, H204R, V223H, T246E, N249D, E286R, Q2911, E302K, E302R, F309N, M320L, P33OE, L435G, L435R, N454K, D524A, D524G, D524N, E562Q, D583N, H594Q, E607K, D653N, and L671P.

[0155] In some embodiments, the reverse transcriptase can also be a wild-type or modified transcription xenopolymerase (RTX), avian myeloblastosis virus reverse transcriptase (AMV RT), Feline Immunodeficiency Virus reverse transcriptase (FIV-RT), FeLV-RT (Feline leukemia virus reverse transcriptase), HIV-RT (Human Immunodeficiency Virus reverse transcriptase). In some embodiments, the reverse transcriptase can be a fusion of MMuLV to the Sto7d DNA binding domain (see lonnidi et al.; https: / / doi.org / 10.! 101 / 2021.11.01.466786).

[0156] Examples of prime editors or gene editors can be found in the following: W02020 / 191153, W02020 / 191171, WO2020 / 191233, WO2020 / 191234, WO2020 / 191239, W02020 / 191241, WO2020 / 191242, WO2020 / 191243, WO2020 / 191245, WO2020 / 191246, WO2020 / 191248, WO2020 / 191249, each of which is incorporated by reference herein in its entirety.2.4. Delivery

[0157] Also provided herein are compositions used to deliver any of the gRNAs, the polynucleotide-programmable nucleotide binding domains, the RNPs, and / or the systems described herein. In some embodiments, the gRNAs, the polynucleotide-programmable nucleotide binding domains, the RNPs, and / or the systems described are delivered using a single modality. In some embodiments, the gRNAs, the polynucleotide-programmable nucleotide binding domains, the RNPs, and / or the systems described herein are delivered using a two or more modalities.

[0158] In some embodiments, the gRNAs, the polynucleotide-programmable nucleotide binding domains, the RNPs, or the systems described herein are delivered using a viral vector. In some embodiments, the gRNAs, the polynucleotide-programmable nucleotide binding domains, the RNPs, and / or the systems described herein are delivered using a lipid nanoparticle (LNP). In some embodiments, the gRNAs, the polynucleotide-programmable nucleotide binding domains, the RNPs, and / or the systems described herein are delivered using a combination of one or more viral vectors and one or LNPs.

[0159] In some embodiments, the viral vector is selected from: an adeno-associated virus (AAV), a lentivirus, an adenovirus, a herpes simplex virus, and a retrovirus.

[0160] Donor sequences can be introduced as a naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV), as described above for nucleic acids encoding a DNA-targeting RNA and / or site-directed modifying polypeptide and / or donor polynucleotide.2.4.1. rAAV

[0161] Also provided herein are recombinant adeno-associated virus (rAAV) used to deliver any of the gRNAs, the polynucleotide-programmable nucleotide binding domains, the RNPs, and / or the systems described herein.

[0162] In some embodiments, a recombinant adeno-associated virus (rAAV) includes: a capsid protein having at least 80% sequence identity to a sequence selected from SEQ ID NO: 212-222, 252-255; and an AAV genome comprising: a polynucleotide sequence encoding a gRNA (e.g., any of the gRNA described in Section 4.1); and a polynucleotide sequence encoding a polynucleotide-programmable nucleotide-binding domain (e.g., any of polynucleotide- programmable nucleotide-binding domains described in Section 4.2.1)

[0163] In some embodiments, the rAAV includes a capsid protein selected from: AAV2, AAV5, AAV6, AAV8, AAV9, AAV-DJ; AAV-KP1, AAV-LK03, AAV- AM, AAV.cc47, AAV.cc47-81, AAV.cc47-82, AAV.cc47-83, AAV.cc47-84, AAV.cc47-85, AAV.cc47-86, AAV.cc47-87,AAV.cc47-88, or AAV.cc47-89.

[0164] Non-limiting examples of capsid proteins that can be used in the rAAV include those described in: U.S. Patent Nos. 8,067,014; 7,588,772; and 9,169,299, and U.S. Patent Publication Nos: 2020 / 0024616, 2021 / 0355481, and 2023 / 0151389, each of which are hereby incorporated by reference in their entireties.

[0165] In some embodiments, the capsid protein is selected from a capsid described in Table 9.

[0166] In further embodiments, David Liu’s 2022 paper (Davis et al., (2022). Nat Biomed Eng, 6:1272-1283) depicted the use of five Cas enzymes for single AAV base editing (PAM, guide RNA length in parenthesis): SaCas9 (NNGRRT, 21-23 nt), SaCas9-KKH (NNNRRT, 21-23 nt), NmeCas9 (NNNNCC, 24 nt), CjCas9 (NNNVRYAC, 22 nt), SauriCas9 (NNGG, 21 nt). SingleAAV plasmids are available from Addgene for SaCas9, SaCas9-KKH, and SauriCas9. However, a paper from Erik Sontheimer (Zhang et al., (2022) GEN Biotechnol, l(3):285-299) used an NmeCas9 single AAV plasmid with adenine base editing. Targeting of the adenine base editing of the forward and reverse strands are depicted in Table X and Table 8. A non-exhaustive list of base editors with their PAM sequence can also be seen in Table Y.

[0167] In some embodiments, where a rAAV is the mode of delivering, the polynucleotide- programmable nucleotide-binding domain is selected from: a base editor, a prime editor, a Cas9, a Cas9 nickase, a dead Cas9, a Casl2a, a Casl2a nickase, a dead Casl2a, a FokI nuclease, an integrase, a zinc finger nuclease, or a transcription activator-like effector nuclease, (see Section 2.3.1). In some embodiments, the polynucleotide-programmable nucleotide-binding domain is a base editor. In some embodiments, the base editor is a cytosine base editor (CBEs), an adenine base editor (ABEs), or non-canonical ABE base editor (see Section 2.3.1). In some embodiments, the base editor is selected from: VQR-BE3, ABEmax-VRQR, NG-ABEmax, ABEmax, xABEmax, xCas9(3.6)-ABE, xCas9(3.7)-ABE, CP-CBEmax, CP-ABEmax, VRQR, NG, xBE, xCas9(3.6)-CBE, xCas9(3.7)-CBE, BE4max, AncBE4max, and ABE8e (see Section 2.3.1). In some embodiments, the polynucleotide-programmable nucleotide-binding domain is Streptococcus pyogenes Cas 9 (SpCas9) or Staphylococcus aureus (SaCas9) (see Section 2.3.1).2.4.2. Nanoparticle Delivery

[0168] Any of the disclosed compositions including, but not limited to potentiating factors, gene editing molecules, polynucleotide-programmable nucleotide-binding domain, etc., can be delivered to the target cells using a nanoparticle delivery vehicle. Non-viral vectors such as nanoparticles have also been investigated for their ability to target the kidney (Peek J.I, et al., (2023). Nature Reviews Nephrology, 19(7): 3-5). Work by Williams et al. in 2016 resulted in the synthesis and characterization of nanoparticles that were able to localize to the renal proximal tubules around 7 times more efficiently compared to other organs (Williams R.M, et al., (2015). Nano Lett, 15(4): 2358-2364).

[0169] In some embodiments, the compositions may be packaged in nanoparticles. For example, in some embodiments, the gene editing technology and / or polynucleotide-programmable nucleotide-binding domain can be incorporated into nanoparticles. In some embodiments, thegene editing technology and / or polynucleotide-programmable nucleotide-binding domain, and the gRNA are packaged in nanoparticles. The different compositions can be packaged in the same nanoparticles or different nanoparticles. For example, the compositions can be mixed and packaged together. In some embodiments, the different compositions can be packaged separately into separate nanoparticles wherein the nanoparticles are similarly or identically composed and / or manufactured. In some embodiments, the different compositions can be packaged separately into separate nanoparticles wherein the nanoparticles are differentially composed and / or manufactured.

[0170] Nanoparticles generally refers to particles in the range of between 500 nm to less than 0.5 nm, preferably having a diameter that is between 50 and 500 nm, more preferably having a diameter that is between 50 and 300 nm. Cellular internalization of polymeric particles is highly dependent upon their size, with nanoparticulate polymeric particles being internalized by cells with much higher efficiency than microparticulate polymeric particles. For example, Desai, et al. have demonstrated that about 2.5 times more nanoparticles that are 100 nm in diameter are taken up by cultured Caco-2 cells as compared to microparticles having a diameter on 1 pM (Desai, et al., Pharm. Res., 14:1568-73 (1997)). Nanoparticles also have a greater ability to diffuse deeper into tissues in vivo.

[0171] Nanoparticles are ideal materials for the fabrication of gene editing delivery vehicles. Nanoparticles aid in the control over the size range of fabrication, down to 100 nm or less, an important feature for passing through biological barriers; reproducible biodegradability without the addition of enzymes or cofactors; capability for sustained release of encapsulated, protected nucleic acids over a period; well-understood fabrication methodologies that offer flexibility over the range of parameters that can be used for fabrication, including choices of the polymer material, solvent, stabilizer, and scale of production; and control over surface properties facilitating the introduction of modular functionalities into the surface.

[0172] The polymer that forms the core of the nanoparticle may be any biodegradable or non- biodegradable synthetic or natural polymer. Natural polymers include alginate and other polysaccharides, collagen, albumin and other hydrophilic proteins, zein and other prolamines andhydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.

[0173] In some embodiments, non-biodegradable polymers can be used, especially hydrophobic polymers. Examples of non-biodegradable polymers include ethylene vinyl acetate, poly(meth) acrylic acid, copolymers of maleic anhydride with other unsaturated polymerizable monomers, poly(butadiene maleic anhydride), polyamides, copolymers and mixtures thereof, and dextran, cellulose and derivatives thereof.

[0174] Other suitable biodegradable and non-biodegradable polymers include, but are not limited to, polyanhydrides, polyamides, polycarbonates, polyalkylenes, polyalkylene oxides such as polyethylene glycol, polyalkylene terepthalates such as poly(ethylene terephthalate), polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyethylene, polypropylene, poly(vinyl acetate), poly vinyl chloride, polystyrene, polyvinyl halides, polyvinylpyrrolidone, polymers of acrylic and methacrylic esters, polysiloxanes, polyurethanes and copolymers thereof, modified celluloses, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt, and polyacrylates such as poly(methyl methacrylate), poly(ethylmethacrylate), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate). These materials may be used alone, as physical mixtures (blends), or as co-polymers, as shown in W02016094880A1.2.4.3. Virus-like Particles (VLPs)

[0175] In certain embodiments, the therapeutic agents delivered by the VLP-based delivery system are nucleic acids. Therapeutic agents that are nucleic acids can be, for example, siRNA, gRNA, or shRNA molecules or plasmids encoding them. Other therapeutic agents can be, for example, small molecules, such as small molecules with anti-viral or anti-cancer activity.

[0176] C AS-containing VLPs may be prepared from a variety of virus-derived particles, and especially with virus-derived particles wherein the guide RNA (SEQ ID NO: 8) and base editor(ABE8e) contained therein may originate from a variety of viruses. Notably, it is described in the examples CAS-containing virus-derived particles comprising a guide RNA (SEQ ID NO: 8), as well as CAS-containing virus-derived particles comprising ABE8e base editor. It is shown herein that both kinds of CAS-containing virus-derived particles efficiently engineer a targeted gene, e.g., efficiently cleave a targeted gene.

[0177] Further, the ABE8e base editor-containing virus-derived particles efficiently alter desired target sequences in vivo. The ABE8e base editor-containing virus-derived particles may be used to induce desired genomic alterations (e.g. induce a cleavage at a desired location in the genome) in living embryos. It is also known herein that the genomic alterations performed in the living embryos are present in the resulting adult mammal and are then transferred to the subsequent generations.

[0178] It is noted that major gene expression processes (such as transcription and translation) are less active in some primary cells subsets that may be major targets for CRISPRs strategies and could therefore decrease the efficiency of conventional delivery methods like DNA transfection and conventional lentiviral vector.

[0179] In this regard, the Cas9-virus-derived particles appears as a tool of choice for genome editing, especially for genome editing in non-activated, non-dividing primary cells like lymphocytes, which poorly support transfection / transduction procedures, and display a low metabolism prior activation.

[0180] The Cas-containing virus-derived particles described herein, especially the Cas9- containing virus-derived particles, may be easily produced in large amounts in the absence of gRNAs, to obtain VLP batches that are carefully dosed and quality-controlled. These Cas9-VLPs can later be combined in a custom-dependent manner with gRNA(s)-containing vesicles and / or targeting nucleic acid-containing vesicles, so as to complement the system by specific gRNAs or specific reparation template, or both.

[0181] As used herein, a virus-derived particle means a particle formed from the assembly of viral structural proteins which are associated so as to form the particle core that will be later enveloped with a membrane, (which virus-derived particle does not contain any nucleic acidencoding a nucleic acid or a protein of interest). Thus, in contrast to most of the virus-derived particles known in the art, which are designed for delivering expression nucleic acids in the transduced cells, a virus-derived particle as described herein is designed for delivering proteins, and optionally non-coding nucleic acids in the transduced cells, i.e. at least a Cas protein. A virus-derived particle according to the invention may also contain one or more non-coding nucleic acids, which non-coding nucleic acids encompass CRISPR-Cas system guide RNA(s) and targeting nucleic acids. It may arise that a virus-derived particle as described herein may contain traces of coding nucleic acids originating from the cells that are used for producing them, such as traces of mRNAs or plasmid DNA originating from the said producing cells. The small amount of coding nucleic acids that may in some occasions be present within the virus-derived particles are generally passively encapsulated. However, it shall be clearly understood that the virus-derived particles described herein are not at all dedicated to transport any coding nucleic acid of interest but, as described in detail throughout the entire specification, these virus-derived particles are in contrast only dedicated to transport proteins, mainly one or more proteins having a Cas endonuclease activity, and in some embodiments also non-coding nucleic acids of interest, namely (i) one or more CRISPR-guide RNA(s) and / or one or more targeting nucleic acid(s). Retrovirus vectors suitable for producing the virus-derived particles described herein allow (1) transfection of the packaging vectors and envelope vectors into the host cell to form a packaging cell line that produces the virus-derived particles essentially free from packaging vector RNA, and (2) the packaging of the Cas protein and optionally also of the CRISPR guide RNA(s) and eventually of a targeting nucleic acid into the virus-derived particles, as described in WO20 17068077.

[0182] In another aspect, the invention provides a vaccine containing a polynucleotide encoding the VLP of any previous aspect or any other VLP delineated herein. In one embodiment, the vaccine is a DNA vaccine.2.4.4. Extracellular vesicles

[0183] This disclosure provides methods of using an extracellular vesicle (e.g., exosome), such as any of the vesicles described herein, to deliver one or more exosomal cargos to a recipient cell. For example, an exosome can be administered to a mammal (e.g., a human) to alter the gut microbiome of a mammal, to regulate (e.g., increase or decrease) the immune response of amammal, to enhance the fertility of a mammal, to alter (e.g., increase or decrease) the metabolism of a mammalian cell, to alter (e.g., increase or decrease) the gene expression of a mammalian cell, to increase the muscle strength of a mammalian cell, to enhance neurological processes of a mammal, and / or to treat a mammal having a disease. In some embodiments, an exosome can be loaded with an exogenous cargo (e.g., a therapeutic agent) and used to deliver the exogenous cargo to a mammalian receptor cell.

[0184] Extracellular vesicles that can be used to encapsulate or carry one or more cargos as described herein include a biological membrane (e.g., a lipid bilayer) that surrounds a lumen. Any appropriate extracellular vesicle can be used as described herein. Examples of extracellular vesicles include, without limitation, exosomes, microvesicles, oncosomes, ectosomes, prostasomes, matrix / calcifying vesicles, tolerosomes, cardiosomes, and vexosomes. Extracellular vesicles and their respective properties are discussed elsewhere (see, e.g., Lotvall et al. 2014 Journal of Extracellular Vesicles 3:26913; and Zempleni et al., 2013 Nature Reviews Drug Discovery 12:347-357). In some embodiments, an exosome can be used to carry or encapsulate one or more cargos as described herein. In some embodiments, an exosome comprises a biological membrane surrounding a lumen, glycoprotein(s) embedded within the biological membrane such that one or more glycans on the glycoprotein(s) are presented on the outer surface of the biological membrane, and cargo encapsulated in the lumen, as discussed in WO2018170332.2.4.5. Cell based delivery

[0185] Cell-based vehicles are particularly attractive for delivery of bio-therapeutic agents that are difficult to synthesize, have reduced half-lives, limited tissue penetrance or are rapidly inactivated upon direct in vivo introduction. The cell-based delivery system possesses a number of advantages including prolonged delivery times, targeting of drugs to specialized cell compartments and biocompatibility. The use of a physiological carrier to deliver therapeutics throughout the body to both improve their efficacy while minimizing inevitable adverse side effects, is an appealing perspective that can be applied in many clinical settings.

[0186] In a related aspect, the present disclosure relates to a method for the delivery of a cellbased therapeutic agent to an organ or tissue, the cell-based repair of which is desired. Themethod comprises flushing an organ or tissue in which the treatment is desired with a buffered physiological solution to remove blood and blood products; establishing the organ or tissue in a perfusion system comprising a warm non-blood perfusion solution, where the perfusion system is capable of restoring oxidative metabolism in the organ or tissue and maintaining the organ or tissue in a near-normal metabolic state at 25°-37° C; and contacting the organ or tissue with a cell-based therapeutic agent by introducing it via the recirculating perfusion solution.

[0187] In a related aspect, the present disclosure relates to a method for the delivery of a cellbased therapeutic agent to an organ or tissue, the cell-based repair of which is desired. The method comprises flushing an organ or tissue in which the treatment is desired with a buffered physiological solution to remove blood and blood products; establishing the organ or tissue in a perfusion system comprising a warm non-blood perfusion solution, where the perfusion system is capable of restoring oxidative metabolism in the organ or tissue and maintaining the organ or tissue in a near-normal metabolic state at 25°-37° C.; and contacting the organ or tissue with a cell-based therapeutic agent by introducing it via the recirculating perfusion solution. In some embodiments of the invention, an AAV virus containing the guide RNA and CAS protein is delivered in a perfusion solution. In other embodiments, an LNP containing the guide RNA and CAS protein is delivered in a perfusion solution. In some embodiments, a VLP containing the guide RNA and CAS protein is delivered in a perfusion solution.2.4.6. LNP Delivery

[0188] Also provided herein are lipid nanoparticles used to deliver any of the gRNAs, the polynucleotide-programmable nucleotide binding domains, the RNPs, and / or the systems described herein.

[0189] In some embodiments, a lipid nanoparticle (LNP) includes: a polynucleotide sequence encoding a gRNA (e.g.., any of the gRNA described in Section 4.1); and a polynucleotide sequence encoding a polynucleotide-programmable nucleotide-binding domain (e.g., any of the polynucleotide-programmable nucleotide-binding domains described in Section 4.2.1).

[0190] In some embodiments, a lipid nanoparticle (LNP) includes: a ribonucleoprotein complex(RNP) comprising a gRNA (e.g.., any of the gRNA described in Section 4.1) and apolynucleotide-programmable nucleotide-binding domain (e.g., any of the polynucleotide- programmable nucleotide-binding domains described in Section 4,2,1),

[0191] In some embodiments, where an LNP is the mode of delivering, the polynucleotide- programmable nucleotide -binding domain is selected from: a base editor, a prime editor, a Cas9, a Cas9 nickase, a dead Cas9, a Casl2a, a Casl2a nickase, a dead Casl2a, a FokI nuclease, an integrase, a zinc finger nuclease, or a transcription activator-like effector nuclease, (see Section 2.3.1). In some embodiments, the polynucleotide-programmable nucleotide-binding domain is a base editor. In some embodiments, the base editor is a cytosine base editor (CBEs), an adenine base editor (ABEs), or non-canonical ABE base editor (see Section 2.3.1). In some embodiments, the base editor is selected from: ABEmax-VRQR, ABEmax-NG, ABEmax, xABEmax, xCas9(3.6)-ABE, xCas9(3.7)-ABE, CP-CBEmax, CP-ABEmax, BE3-xCas9-3.6, BE3-xCas9-3.7, BE4max-xCas9(3.6), BE4max-xCas9(3.7)-, BE4max, BE4max-CP1028, BE3- pBK-VQR, AncBE4max, CBE6a, CBE6b, CBE6c, CBE6d, ABE8e, ABE8e-NG, ABE8e- CP1028, pCMV-SpRY-ABE8e, ABE8.20-d, ABE8.20-m, ABE8.17-m, ABE8e-CP1041, ABEmax-CP1012, ABE7.10-3.6, ABE7.10-3.7, ABE7.10, and pCAG-CBE4max-SpRY. (see Section 2.3.1). In some embodiments, the polynucleotide-programmable nucleotide-binding domain is Streptococcus pyogenes Cas 9 (SpCas9) or Staphylococcus aureus (SaCas9) (see Section 2.3.1).2.5. Cell

[0192] Also provided herein are engineered cells with increased PKD1 mRNA levels, comprising: a genetic modification in the 3’ UTR of PKD, wherein the engineered cell has increased PKD1 mRNA levels as compared to a cell that does not have a genetic modification in the 3’ UTR of PKD 1.

[0193] In some embodiments, the engineered cell has increased PKD1 mRNA levels of at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 300%, at least 400%, at least 500%, at least600%, at least 700%, at least 800%, at least 900%, or at least 1000% as compared to a cell that does not have a genetic modification in the 3’ UTR of PKD1.

[0194] In some embodiments, the engineered cell has increased PKD1 mRNA levels of at least 0.5-fold, 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10 or more fold as compared to a cell that does not have a genetic modification in the 3’ UTR ofPKDl.

[0195] In some embodiments, the engineered cell has increased PKD1 mRNA levels of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 count per million(cpm) as compared to PKD1 cpms in a cell that does not have a genetic modification in the 3’ UTR ofPKDl.2.6. Methods of Increasing PKD1 mRNA Levels

[0196] Also provided herein are methods for increasing PKD1 mRNA levels in a cell. In some embodiments, a method for increasing PKD1 mRNA levels in a cell includes: introducing into the cell any one of the systems described herein (see Section 2.3.1.6), any one of the rAAVs described herein (see Section 2.4.1), any of the LNPs described herein (see Section 2.4.6), or a combination thereof.

[0197] In some embodiments, a method for increasing PKD1 mRNA levels in a cell includes: introducing into the cell any one of the systems described herein (see Section 2.3.1.6), any one of the rAAVs described herein (see Section 2.4.1), any of the LNPs described herein (see Section 2.4.6), or a combination thereof, whereby, upon introducing the system into a cell, the system modifies a binding site of a miRNA-17 family miRNA in the 3’ UTR of PKD1, thereby preventing binding of the miRNA-17 family miRNA and de-repressing PKD1 mRNA levels.

[0198] In some embodiments, the cell is a kidney cell. In some embodiments, the cell or kidney cell is in vitro or in vivo. In some embodiments, the cell or kidney cell is in vitro. In someembodiments, the cell or kidney cell is in vivo in a subject, where the subject has or is suspected of having a PKD-associated disorder.

[0199] In some embodiments, the method increases PKD1 mRNA in a cell by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% as compared to a cell not subjected to the methods described herein and / or does not have a genetic modification in the 3’ UTR ofPKDl .

[0200] In some embodiments, the method increases PKD1 mRNA in a cell by at least 0.5-fold, 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10 or more fold as compared to a cell not subjected to the methods described herein and / or does not have a genetic modification in the 3’ UTR of PKD1.

[0201] In some embodiments, the method increases PKD1 mRNA in a cell by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 count per million (cpm) as compared to PKD1 cpms in a cell not subjected to the methods described herein and / or does not have a genetic modification in the 3’ UTR of PKD1.2.7. Methods of Treating a PKD-Associated Disorder

[0202] Also provided herein are methods of treating a subject having or suspected of having a PKD-associated disorder.

[0203] In some embodiments, a method of treating a subject having or is suspected of having a PKD-dependent associated disorder includes: introducing into the cell any one of the systems described herein (see Section 2.3.1.6), any one of the rAAVs described herein (see Section 2.4.1), any of the LNPs described herein (see Section 2.4.6), or a combination thereof.

[0204] In some embodiments, a method of treating a subject having or is suspected of having a PKD-dependent associated disorder includes: introducing into the cell any one of the systems described herein (see Section 2.3.1.6), any one of the rAAVs described herein (see Section 2.4.1), any of the LNPs described herein (see Section 2.4.6), or a combination thereof, whereby, upon introducing the system into the subject, the system modifies a binding site of a miRNA-17 family miRNA in the 3’ UTR of PKD1, thereby preventing binding of the miRNA-17 family miRNA and de-repressing PKD1 mRNA levels.

[0205] In some embodiments, the PKD-dependent associated disorder is autosomal dominant polycystic kidney disease (ADPKD).

[0206] In some embodiments, the ADPKD patient has a PKD 1 mutation. In other embodiments, the ADPKD patient has a PKD2 mutation.

[0207] In some embodiments, the method of treating ADPKD will slow or halt the expected decline in glomerular filtration rate, improve cellular trafficking of PKD1 or PKD2 reduce serum creatinine, decrease total kidney volume, reduce blood urea nitrogen, decrease the number of cysts, and reduce cystic area.3. EXAMPLES3.1. Example 1: Proof-of-concept for increasing PKD1 mRNA levels by using genetic modification to prevent binding of the miRNA-17 family miRNA

[0208] This Example outlines a proof-of-concept study for increasing PKD1 mRNA levels by using genetic modification to prevent binding of the miRNA-17 family miRNA. The genetic modification is mediated by a system comprising a gRNA and an editor (also referred to herein as a polynucleotide-programmable nucleotide-binding domain). In particular, this Example describes guide RNA design for CRISPR-Cas editing of the miR-17 binding site in the 3’ UTR of PKD1. Proof-of concept studies are performed in both mouse and human cells. gRNAs and a different CRISPR-Cas systems including base editors, prime editors, and standard CRISPR-Cas editors are contemplated. In a non-limiting example, several Cas nucleases for each type of editor, including SpCas9 (PAM = NG or NGG), SaCas9 (PAM = NNGRRT), and LbCasl2a (PAM = TTTV) are used.3.1.1. gRNA Selection for Human PKD1 3’ UTR

[0209] Twenty-one guide RNAs that target the miR-17 human sequence were identified (see Table 1). These guide RNA sequences were selected based on having a C->T or A->G edit in positions 4-9 starting from the 5’ end of a guide RNA placed immediately upstream of a PAM (NG, NNGRRT, or TTTV). For example, Position 3 corresponds to the third nucleotide in the guide RNA reading from the 5’ direction.3.1.2. System Selection

[0210] The gRNA and system combinations described in Table 8 (also Table 1) (see Section 2.2) have been selected for testing.3.1.3. Introducing gRNA / Polynucleotide-Programmable Nucleotide-Binding Domain

[0211] To introduce the gRNA and system combinations into cells, the gRNA can be cloned into a pmCherry-U6-empty (mCherry) reporter plasmid and co-transfected with a second GFP plasmid carrying the desired gene editor. GFP+ mCherry+ double positive cells can be isolated via fluorescence-activated cell sorting to purify edited cells. In some embodiments, the gRNA will be transfected as a chemically modified guide RNA complexed directly with Cas proteininto a ribonucleoprotein complex. In other embodiments, the gRNA and editing machinery can be packaged and delivered with a viral or nanoparticle vector.3.1.4. Predicted Results

[0212] The systems from Table 8 (see also Tables 1 and 2) that are predicted to achieve the greatest chance of successful, high efficiency knockout include: gRNA8 (BE4max, AncBE4max, ABEmax, ABE8e); gRNA3 and gRNA8 standard SpCas9 CRISPR (PX458); gRNA2 (all non- canonical ABE editors: ABEmax- VRQR, NG-ABEmax, xABEmax, xCas9, etc.), gRNA5, gRNA7, and gRNA9 (all non-canonical ABE and CBE editors).

[0213] Overall, this proof-of-concept is expected to show that a system comprising a gRNA / polynucleotide-programmable nucleotide-binding domain designed to target and induce a genetic modification in the binding site of miR-17 in the 3’ UTR of PKD1.3.1.5 Example 2: Design of gRNAs

[0214] For the generation of gRNAs, as seen in Table 1, the PAM sites flanking the target region, the specific modification window, and any off-target effects were taken into consideration. Many of the gRNA constraints are dictated by the editing tools available for use as the proteins themselves dictate the PAM site. Due to PAM site requirements, not all gRNAs can be used with each type of base editor, but combining both a variety of editors and gRNAs allows for various possible editing systems to be tested. To ensure all editor + gRNA combinations are fully explored, gRNAs were tested with all ABEs and / or CBEs, canonical or non-canonical, despite mismatches between PAM site requirements and availability. Additionally, both the first and second A or C in the target region, detailed for each guide in Table 8, was assessed for editing to standardize comparisons with the only exception being gRNAl, as the target region only has one C.3.1.6 Example 3: Canonical base editors

[0215] One method of introducing plasmid DNA into mammalian cells is through electroporation, the use of a high-voltage electric shock that disrupts the cell membrane creating pores that allow macromolecules to pass into the cell (Potter, H, et al., (2003). Curr Protoc MolBiol, Chapter 9, 9.3). This technique can be successfully applied to a wide variety of cell lines with varying efficiency based on cell type, the macromolecule being introduced, and various other procedural considerations. Here the Invitrogen® Neon® Transfection System was used with 293T cells (ATCC: CRL-3216). The 293T cells were originally isolated from human embryo kidney tissue and are designed to be a highly transfectable version of the 293 cell line. The cells contain the temperature sensitive gene for the SV40 T-antigen, which assists in production of adeno-associated virus vectors, another common use for the cell line (De, BP, et al., (2023). Hum Gene Ther, 34(15-16): 697-704). They are adherent cells with an epithelial morphology.

[0216] Following cell transfection, it is necessary to determine if the delivery and expression of the editor system is successful, which can be accomplished using fluorescence-activated cell sorting (FACS). This method employs flow cytometry which uses lasers to produce scattered and fluorescent light signals that can be read by detectors and then converted to computer- interpretable signals allowing for cell analysis (McKinnon KM, et al., (2018). Curr Protoc Immunol, 120: 5.1.1-5.1.11). Traditional flow cytometers allow for analysis of the number of cells expressing the fluorescent proteins of interest, while cell sorters (FACS) allow for both analysis and the purification of specific cell populations (McKinnon KM, et al., (2018). Curr Protoc Immunol, 120: 5.1.1-5.1.11). As previously described, cells that receive the full editor system will fluoresce both green and red, allowing for both analysis and sorting to take place. Sorting resulted in enrichment of the cell population that was successfully expressing both the gRNA and base editor plasmids, these cells are termed “double positive” or “DP” cells.

[0217] To assess base editing in the transfected DP cells, it is necessary to obtain purified cellular DNA. The method of DNA purification varied based on the number of cells present, as samples with higher cell numbers can undergo more vigorous purification, whereas samples with lower cell counts require more refined procedures designed to optimize DNA yield. The purification method does not modify the downstream use of the purified DNA.

[0218] For samples with a greater number of cells, generally exceeding 100,000, the ZymoQuick-DNA Miniprep Plus Kit (Zymo Research: D4068) was followed according to manufacturer protocols. Prior to beginning the Zymo instructions the cells were spun down at17,000 g for 5 minutes and the supernatant was carefully aspirated to avoid disturbing the cell pellet. DNA was eluted in 50 uL of elution buffer and the purified DNA was quantified using the Thermo Scientific™ NanoDrop™ One Microvolume UV-Vis Spectrophotometer (ThermoF isher : 13 -400-519).

[0219] For samples containing a lower concentration of cells QuickExtract (Biosearch Technologies: SS000035-D2) was used. The tubes were first spun down at 17,000 g for 5 minutes, the supernatant was aspirated fully being careful not to disturb the pellet, the pellet was then resuspended in 50 uL of QuickExtract and transferred to a PCR tube. The samples were vortexed for ~15 seconds and placed in a thermocycler to incubate at 65 °C for 6 minutes, they were then removed from the thermocycler and vortexed for ~15 seconds. Next, they were placed back into the thermocycler and incubated at 98°C for 2 minutes, cooled to 4°C, and vortexed a final time for ~15 seconds, at this point they were ready for downstream applications.

[0220] Although the purified genomic DNA contains the region of interest for the guided base edits, it was not feasible to carry out full genome sequencing for all samples. Additionally, as there is only one region of interest, this would generate unnecessary data. To solve this issue, polymerase chain reaction (PCR) was carried out to amplify only the region of interest which was then sequenced using Sanger Sequencing. Sanger sequencing was carried out in collaboration with Azenta LifeSciences, the unpurified PCR products were sent to Azenta for sequencing. Azenta retained the primers used for PCR at their facility and used the same primers for Sanger sequencing. The primers specify the region of DNA to be amplified, and heating and cooling the PCR reaction to specified temperatures facilitates denaturing of the template DNA, annealing of the primer oligos, extension of the fragment region by DNA polymerase, and enzyme inactivation. This results in the amplification of a specific region of interest in the genome (McKinnon KM, et al., (2018). Curr Protoc Immunol, 120: 5.1.1 -5.1.11). PCR is a powerful tool for genetic analysis and is pivotal in this work as it was a precursor for Sanger sequencing.3.1.7 Example 4: Editing verification

[0221] To examine the genome edits, it is necessary to sequence the PCR products obtained following transfection and DNA isolation. The sequencing was carried out by Azenta LifeSciences using Sanger sequencing. Sanger sequencing takes advantage of the catalytic mechanism of DNA polymerase and provides fluorescently labeled dNTPs that lack the 3’- hydryoxyl needed for continued DNA synthesis (Dey, P, et al., (2022). Springer Nature Singapore, 247-261). When these labeled dNTPs were incorporated for synthesis, they cause chain termination and will emit a fluorescent signal, with each of the four bases having a different fluorophore present synthesis (Dey, P, et al., (2022). Springer Nature Singapore, 247- 261). Examining the patterning of these fhiorophores allows the full DNA sequence of the region of interest to be determined.

[0222] The initial set of experiments with the base editor system used the canonical NGG PAM site with a total of four different editors and one gRNA. Of the gRNA chosen, gRNA8 had the highest likelihood of editing in the target window. Neon transfections were carried out according to the workflow depicted in FIG. 1A, resulting in FACS plots (FIG. IB), and Sanger sequencing data (FIG. 1C) for each sample. Representative data for ABE8e + gRNA8, the initial lead candidate, is shown. The cumulative findings from these initial experiments are depicted in FIG. ID through an examination of the percent editing at the most efficiently modified base in the target window. Importantly, the percent editing efficiencies examined were obtained from double positive cells only, meaning the cells were expressing both the base editor and gRNA expression plasmids, as shown by their positive mCherry and GFP fluorescent signals.

[0223] The initial testing showed promising results for the combination of ABE8e + gRNA8. The wide toolbox of base editors, both ABEs and CBEs, and numerous gRNAs with the potential to cause edits in the target region warranted further exploration to ensure the ideal combination of editor + gRNA would be used for downstream applications. Importantly, many of the gRNAs required non-canonical editors due to PAM site availability, and many of these editors do not have GFP in their plasmid form.3.1.8 Example 5: Next-generation sequencing data

[0224] To prepare the samples for NGS, DNA was first collected as detailed above using either the Zymo Quick-DNA Miniprep Plus kit or QuickExtract and quantified. Each DNA sample was then diluted to a concentration of 50 ng / uL to normalize the amount of PCR product obtained. A total of 8 PCR reactions per sample were then carried out by combing 12.5 uL of NEBNext®Ultra™Q5® Master Mix (NEB: M0544S), 1 uL of a 10 uM stock of primer #22, 1 uL of a 10 uM sock of primer #23, 9.5 uL of molecular grade water, and 1 uL of DNA template, assuming normalization. If DNA concentration was below 50 ng / uL the volume was increased so 50 ng would be added, and the volume of water was decreased accordingly as to not exceed 25 uL total. The reactions were prepared on ice and then placed in a thermocycler running the following program: 98°C for 30 seconds, 30 cycles of the following [98°C for 30 seconds, 63°C for 15 seconds, 72°C for 15 seconds], 72°C for 2 minutes, and hold at 4°C. To confirm amplification 3 uL of PCR product was run on a 1.5% agarose gel as detailed previously. Half of the replicate PCR reactions per sample (4 total) were then pooled resulting in a total of 2 replicates per sample. The volume of the pooled samples was then brought to 100 uL using sterile water. Next the MagJET NGS Cleanup and Size Slection Kit (ThermoFisher: K2828) was used according to manufacturer instructions to purify the PCR products. Following purification, the products were again pooled, and the samples were quantified using the Qubit™ lx dsDNA HS Assay Kit (ThemoFisher: Q22320) according to manufacturer instructions with 2 uL of each sample being used for quantification. The fluorometer used here was the Quibt™ 4 Fluorometer (ThermoFisher: Q33238). Following quantification, 500 ng of DNA at a concentration of 20 ng / uL was sent to Azenta Life Sciences for NGS analysis.

[0225] The ABE editors were screened first, prompted by the initial promising results from ABE8e + gRNA8. The pivot to the lipofectamine system has the desired effect, with significantly higher transfection efficiencies being seen in the cells as shown by the fluorescent microscopy images in FIG. 3B. Increased transfection efficiency allowed for editing percentages to be compared between different treatments without the need for FACS. As these screens include no enrichment of DP cells, the editing efficiencies seen were significantly lower than was observed in the Neon transfections (FIG. 2).

[0226] To optimize the lipofectamine system, a 48-well plate was seeded with 293Ts at a density of 37,500 cells / well one day before transfection. On the day of transfection, a wide variety DNA and lipofectamine reagent concentrations were tested to determine what was optimal for the system and the transfection was carried out according to manufacturer protocol (ThermoFisher: L3000001). Approximately 24 hours after the transfection the cells were prepared for flow cytometry analysis. The medium from the wells was aspirated, the cells were washed with PBS,0.5 mL of TrypLE (ThermoFisher: 12604013) was added and allowed to incubate for 10 minutes at 37°C to detach the cells. The TrypLE was then quenched using 1 mL PBS, the cells were transferred to a 1.5 mL Eppendorf tube and spun down at 300 g for 4 minutes. The supernatant was aspirated, the cells were resuspended in 100 uL ethylenediaminetetraacetic acid solution (Sigma: E8008), and transferred to a 96-well plate and kept on ice until the flow. The plate was then run using either the NovoCyte Penteon or Quanteon in collaboration with the Columbia University Flow Cytometry Core and the resulting data was obtained.

[0227] Following the optimization, the resulting conditions were extrapolated to both 6-well and 48-well plate layouts and modified to include 72-hours of incubation with a medium change after 24-hours. This change was critical as 24 hours is sufficient for fluorescent protein expression but is not sufficient for base editing to take place. The specific reaction conditions used are detailed in supplementary data 3. Following transfection, the cells were imaged using an EVOS M7000 (ThermoFisher: AMF7000) imager and the DNA was extracted using the previously described methods. The samples then underwent PCR amplification and sequencing as previously described. For a subset of experiments flow analysis was conducted using the ThermoFisher Attune NxT Flow Cytometer.

[0228] In addition to completing the ABE screen, CBE candidates were also tested with all possible gRNAs to investigate if they would be able to outperform the lead ABE candidates (FIG. 3C / D). The screen demonstrated that the CBEs were unable to successfully edit the first available base in the target region. Three candidates, AncBE4Max, BE4-Max, and CP 1249- CBE4Max combined with gRNA8 were able to edit the second base in the target region more efficiently than ABE8e-CP1028 + gRNA8. Yet, as the end goal is to inhibit miR17 binding to the target region having more than one mismatch, would likely be an advantage, suggesting that the ABE editors remained stronger overall candidates.

[0229] The initial ABE and CBE screens for additional editors were tested. First additional ABEs were tested which were derived through directed evolution of previous adenine base editors (Gaudelli NM, et al., (2020). Nature Biotechnol, 28(7):892-900). Although these editors performed well (FIG. 4B), ABE8e and ABE8e-CP1028 + gRNA8 still showed more efficientediting at both the first and second available base in the target region, suggesting they should remain the lead candidates moving forward.

[0230] Next, a new class of editors was tested which require only a single-nucleotide pyrimidine as their PAM allowing for more editing sites to become available (Huang TP, et al., (2023). Nat Biotechnol, 41 (1):96- 107). The new ABEs, as seen in FIG. 4B, were able to edit with different gRNAs than previously seen due to their more lenient PAM requirements. gRNA9, which is a single base downstream of gRNA8, demonstrated higher editing efficiencies at both the first and second A in the target region with the pCMV-spRY-ABE8e editor, performing 0.4% better than ABE8e + gRNA8 at the first site and 3.1% better than ABE8e-CP1028 + gRNA8 at the second site.. Yet, there are important regulatory concerns to be taken into consideration regarding the less-specific PAM site of this candidate and its potential for off-target editing.

[0231] To follow up on the initial CBE screen, newly published CBEs, derived through phage assisted evolution were also screened (Zhang, E, et al., (2024). Nat Commun, 15(1): 1697). These screens were done against the lead candidates at the time, ABE8e + gRNA8 and ABE8e- CP1028 + gRNA8 (FIG. 5A). These results demonstrated that the CBE6s still struggled to edit the first available base in the target region but were able to edit the second base with higher efficiency than previously seen, allowing them to emerge as potential lead CBE candidates. Akin to the ABEs, a new CBE variant that requires only a single-nucleotide PAM site was also recently made available and was tested to examine its efficiency (FIG. 5B). It demonstrated higher than previously seen editing at the first base in the target region using gRNAl, a reverse strand gRNA.

[0232] Comparison of the editing efficiencies at the different target bases available for the CBEs and ABEs and gRNAs can allow for promising candidates in terms of absolute editing examined. It is also advantageous to identify lead candidates for each site in the target region, as modifying each base will likely have a different impact on miR-17 binding. To assess this, the most promising candidates for base editing of the target region, were mapped onto the target region with their editing efficiency graphed as a value normalized to ABE8e + gRNA8 from the corresponding transfection, as shown in FIG. 6. This view of the target region demonstrates thatfor half of the bases present, efficient editing systems have successfully been identified with lead ABE candidates being able to modify two bases at once.

[0233] The coverage of this region suggests that one of these candidates will likely be able to successfully induce edits that prevent miR17 binding to the target region, leading to the possibility of increased polycystin 1 production, correcting the ADPKD phenotype. To further confirm the increase of polycistin 1 production, a western blot is performed to validate the corrected ADPKD phenotype.3.1.9 Example 6: AAV Delivery in vivo

[0234] Human ADPKD gene therapy development via anti-miR-17 also requires an efficient delivery system for CRISPR-Cas base editing. Initial drug testing was conducted in a mouse model of the disease, as mice were selected as the model organism to analyze AAV delivery. The in vivo experiments were carried out using J AX C57BL / 6J mice (RRID:IMSR_JAX:000664). Injections of and care for the mice were carried out in collaboration with the Columbia University Institute of Comparative Medicine.

[0235] As depicted in FIG. 7 A, mice were injected with AAVs, and their tissues were harvested to assess delivery efficiency. Importantly, AAVs were engineered to express mCherry when successfully delivered to cells, so we assessed delivery using fluorescence microscopy imaging of the tissue slides. FIG. 7B demonstrates the initial findings in all mice from the in vivo #14 experiment, including images of the kidneys, the target tissue, and liver, commonly targeted by all drugs, taken at lOx magnification at fixed exposure times for both brightfield, and mCherry filters. These initial images suggest that AAV(S2) delivers the most efficiently to both the kidney and liver, outperforming AAV9. To further examine the delivery efficiency of each AAVs tested, AAV9, AAV(Sl), and AAV(S2), 40x magnification images were taken of kidney tubule cells at fixed exposures. These focused images, as seen in FIG. 8, further support the initial observation that AAV(S2) provided the most efficient delivery. Furthermore, they highlight that not only does AAV(S2) outperform AAV9, but AAV(Sl) also appears to have more efficient delivery. These encouraging results demonstrate a potential kidney-targeting mechanism that would allow for delivery of the base editing system.

[0236] AAV-mediated miR-17 base editing delivery to the kidney in vivo with a minimum level of efficiency is necessary to achieve a therapeutic benefit in vivo through PKD1 mRNA increase. FIG. 8B shows a quantification of in vivo tubular cell delivery with the AAV(Sl) and AAV(S2) capsids, which transduce 20-40% of kidney tubules in vivo. These capsids are significantly more efficient than AAV9, which make them better candidates for in vivo AAV-mediated miR-17 base editing therapy.

[0237] The data presented here examine both a human base-editing system with the potential to modulate miR17 binding to the 3 ’-UTR oYPkdl and an AAV delivery system. These two components are critical to enable further development of a gene therapy system for ADPKD. Luciferase assays are able provide information regarding the amount of gene product that is produced in a designed system (Carter, M, et al., (2015). Academic press, 311-343). Specifically, the assay can confirm that the base edits being made are modifying miR17 binding to the 3’- UTR. FIG 7 shows promising derepression via luciferase assay for mutants of the miR-17 binding domain, suggesting that binding would be inhibited by the mismatches introduced through base editing. This assay aids in the confirmation that the base edits increase PCI.3.1.10 Example 7: Luciferase assay for miR-17 binding

[0238] A luciferase assay can be performed to confirm that base edited mutants of the miR-17 binding region in the PKD1 3’ UTR increase PCI mRNA and protein levels. In this assay, a plasmid containing the luciferase gene upstream of the PKD1 3’ UTR (SEQ ID NO: 11) is transfected into human kidney epithelial cells along with a microRNA mimic. The microRNA mimic is either miR-17 mimic or a negative control scrambled mimic. In some embodiments, this cell line being transfected is HEK293T, WT9-7, or WT9-12 cells. If this luciferase assay works as planned, one will observe a decrease in luciferase expression in cells treated with miR- 17 mimic compared to scrambled control following lipid-based transfection of the plasmid containing the wildtype miR-17 binding region. If mutations to the miR-17 binding region are successful in preventing PKD1 repression of miR-17, then plasmids transfected with those mutants will not show a decrease in luciferase expression. FIG. 11 shows the results of this luciferase binding assay at 1 sec and 3 sec exposure times and confirms that most mutations to the miR-17 binding region prevent luciferase repression. FIG. 11 also shows the most efficientbase editor and guide RNA combinations that were identified for creating each mutant in the miR-17 binding region. Only two of the mutants from the luciferase assay show statistically significant luciferase repression and these mutants are the most difficult to edit with base editing, which is significant because it means that we can edit all of the significant bases in the miR-17 binding region with the guide RNAs and base editors described in this patent.3.1.11 Example 8: Dual and single AAV base editing of miR-17 binding region

[0239] One example of AAV delivery of base editors is a dual AAV split-intein system. The size of SpCas9, adenine deaminase, and guide RNA is greater than the efficient packaging capacity of AAV (~4.7 kb), so the goal of the split-intein system is to adapt AAV for base editing delivery. In this system, the guide RNA is packaged into one AAV with the first half of the base editor. The second half of the base editor is packaged into a second AAV. Both AAVs combine in cells to form the full base editor using trans-splicing inteins also encoded in each AAV. FIG 12 demonstrates the efficiency of this system in multiple human kidney epithelial cell lines, including HEK293T, WT 9-7, and WT 9-12. Editing efficiencies upwards of 80% are achieved using ABE8e base editor and gRNA8 (SEQ ID NO: 8).

[0240] In addition, although SpCas9, deaminase, and guide RNA exceed the packaging limit of AAV, smaller Cas variants have been engineered that allow for packaging of base editors into a single AAV. Some of these smaller Cas variants include CjCas9, NmeCas9, and SauriCas9 (Davis et al. 2022, Nature Biomedical Engineering). FIG. 14 demonstrates the editing efficiency editing with SauriCas9-ABE8e + gRNA20 (SEQ ID NO: 19) or gRNA21 (SEQ ID NO: 20) compared to SpCas9-ABE8e + gRNA8 (SEQ ID NO: 8) in human kidney epithelial cells using lipid-based transfection of plasmids encoding the guide RNAs and corresponding base editors. These cells were unsorted, so the true editing efficiencies are higher. However, the efficiency of SauriCas9-ABE8e is comparable to SpCas9-ABE8e, suggesting that single AAV editing with gRNA20 (SEQ ID NO: 19) and gRNA21 (SEQ ID NO: 20) is comparable in efficiency to the dual AAV editing shown with SpCas9 and gRNA8 (SEQ ID NO: 8) in FIG. 12. The most efficient guide RNA for SauriCas9 editing was gRNA21 (SEQ ID NO: 20), which is very similar to gRNA8 (SEQ ID NO: 8) as shown in FIG. 9 so these results are consistent. FIG. 9 also shows the layout of all other guide RNAs targeting the miR-17 binding region.3.1.12 Example 9: Increasing PKD1 mRNA levels with AAV-mediated miR-17 base editing.

[0241] FIG. 10 describes the mechanism by which modifying the miR-17 binding region in the PKD1 3’ UTR can increase PKD1 mRNA levels to slow polycystic kidney disease progression. In one example, delivery of a base editor in either a single AAV system or a dual AAV system with trans-splicing inteins induces an edit in the miR-17 binding region and this edit causes an increase in PKD1 mRNA levels. FIG. 13 shows a 4-fold increase in PKD1 mRNA levels using the delta delta Ct quantification method for RT-qPCR following AAV-mediated delivery of ABE8e and gRNA8 (SEQ ID NO: 8) in human kidney epithelial cells. The increase in PKD1 mRNA following treatment with ABE8e + gRNA8 (SEQ ID NO: 8) compared to non-treated cells is statistically significant. ACTB and GAPDH act as housekeeping genes.3.1.13 Example 10: Increasing PCI protein levels with ribonucleoprotein-mediated Cas9 delivery.

[0242] In one example, Cas9 editors targeting the miR-17 binding region can be delivered with ribonucleoprotein. Cas9 protein (20 micromolar, either SaCas9 or SpCas9) is incubated with chemically modified guide RNAs from Synthego at a 9:1 molar ratio of gRNA: protein for 10 minutes at room temperature to create the ribonucleoprotein. In some embodiments, the guide RNA is gRNA3 (SEQ ID NO: 3). In some embodiments, the guide RNA is gRNA8 (SEQ ID NO: 8). The ribonucleoprotein is transfected into human kidney epithelial cells using electroporation. In some embodiments the Thermo Fisher Neon device is used for electroporation with settings of 1150V, 20 ms, 2 pulses. In other embodiments, the settings are 1300V, 30 ms, 1 pulse. In some embodiments, the amount of cells used in 150,000 cells per electroporation. In some embodiments, the amount of cells used in 200,000 cells per electroporation. In some embodiments, the amount of cells used in 2,000,000 cells per electroporation. Cells were harvested 72 hours after electroporation and DNA was extracted to measure the percentage indel formation. Indel quantification is done via next generation sequencing. FIG. 15 shows the percent indel formations in the WT 9-7 immortalized human kidney epithelial cell line using untreated, SpCas9 + gRNA3 (SEQ ID NO: 3) and SpCas9 + gRNA8 (SEQ ID NO: 8) conditions. These indel formation percentages only include indels thatdisrupt the miR-17 binding region, other indels are excluded. FIG. 15 also shows a statistically significant increase in PC 1 protein level via Western Blot for human kidney epithelial cells treated with SpCas9 + gRNA3 (SEQ ID NO: 3) ribonucleoprotein. The increase for SpCas9 + gRNA8 is not statistically significant; however, the percent indel formation is also much lower.Ponceau S stain is used to normalize the total protein loaded per well.

[0243] FIG. 16 shows the indel induction efficiencies of SaCas9 ribonucleoprotein delivery in human kidney epithelial cells. In this example, SpCas9 + gRNA8 (SEQ ID NO: 8) is used as a positive control and generates indels in as high as 60% of cells. In contrast, the SaCas9 indel efficiencies are <10%. Other SaCas9 variants were also tested such as SaCas9-KKH to confirm these results. SaCas9-KKH plasmid (Addgene: 70708) was transfected into human kidney epithelial cells via lipid-based transfection. SaCas9 and SaCas9-KKH show much lower indel induction efficiencies than SpCas9 in this example.EQUIVALENTS AND INCORPORATION BY REFERENCE

[0244] All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequences or GenelD entries), patent application, or patent, was specifically and individually indicated incorporated by reference in its entirety, for all purposes. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.F.R. §1.57(b)(1), to relate to each and every individual publication, database entry (e.g., Genbank sequences or GenelD entries), patent application, or patent, each of which is clearly identified in compliance with 37 C.F.R. § 1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.

[0245] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it is understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. A guide RNA (gRNA) comprising: a spacer sequence complementary to a genomic site in 3’ UTR of PKD1; and a scaffold capable of associating with a polynucleotide-programmable nucleotide- binding domain and guiding the polynucleotide-programmable nucleotide-binding domain to the genomic site in the 3’ UTR of PKD1.

2. A guide RNA (gRNA) comprising: a spacer sequence complementary to at least 17, 18, 19, or 20 contiguous nucleotides of the sequence of SEQ ID NO: 11; and a scaffold capable of associating with a polynucleotide-programmable nucleotide- binding domain and guiding the polynucleotide-programmable nucleotide-binding domain to a sequence complementary to the spacer sequence.

3. A guide RNA (gRNA) comprising: a spacer sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 1-10, 12-20, 244, 245; and a scaffold capable of associating with a polynucleotide-programmable nucleotide- binding domain and guiding the polynucleotide-programmable nucleotide-binding domain to a sequence complementary to the spacer sequence.

4. The gRNA of any one of claims 1-3, further comprising the components of a prime editing guide RNA.

5. The gRNA of any one of claims 1-4, further comprising one or more modified nucleotides.

6. The gRNA of any one of claims 1-5, wherein the gRNA is a synthetic gRNA.

7. The gRNA of any one of claims 1-6, wherein the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 8.

8. The gRNA of any one of claims 1-6, wherein the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO:3.

9. The gRNA of any one of claims 1-6, wherein the spacer comprises a sequence having at least 80% sequence identity to SEQ ID NO: 2.

10. A system for inhibiting a miRNA-17 family miRNA from binding to the 3’ UTR ofPKD1, the system comprising: a gRNA of any one of claims 1-9; and a polynucleotide-programmable nucleotide-binding domain or a polynucleotide encoding the polynucleotide programmable nucleotide-binding domain.

11. The system of claim 10, wherein, upon introducing the system into a cell, the system genetically modifies a binding site of a miRNA-17 family miRNA in the 3’ UTR of PKD1, thereby preventing binding of the miRNA-17 family miRNA and de-repressing PKD1 mRNA.

12. The system of claim 10 or 11, wherein the polynucleotide-programmable nucleotide- binding domain is selected from: a base editor, a prime editor, a Cas9, a Cas9 nickase, a dead Cas9, a Casl2a, a Casl2a nickase, a dead Casl2a, a FokI nuclease, an integrase, a zinc finger nuclease, or a transcription activator-like effector nuclease.

13. The system of any one of claims 10-12, wherein the polynucleotide-programmable nucleotide-binding domain is a base editor.

14. The system of claim 13, wherein the base editor is a cytosine base editor (CBEs).

15. The system of claim 13, wherein the base editor is an adenine base editor (ABEs).

16. The system of claim 13, wherein the base editor is a non-canonical CBE base editor or a non-canonical ABE base editor.

17. The system of claim 13, wherein the base editor is selected from: ABEmax-VRQR, ABEmax-NG, ABEmax, xABEmax, xCas9(3.6)-ABE, xCas9(3.7)-ABE, CP-CBEmax, CP-ABEmax, BE3-xCas9-3.6, BE3-xCas9-3.7, BE4max-xCas9(3.6), BE4max- xCas9(3.7)-, BE4max, BE4max-CP1028, BE3-pBK-VQR, AncBE4max, CBE6a, CBE6b, CBE6c, CBE6d, ABE8e, ABE8e-NG, ABE8e-CP1028, pCMV-SpRY-ABE8e, ABE8.20-d, ABE8.20-m, ABE8.17-m, ABE8e-CP1041, ABEmax-CP1012, ABE7.10-3.6, ABE7.10-3.7, ABE7.10, and pCAG-CBE4max-SpRY.

18. The system of any one of claims 10-17, wherein the polynucleotide-programmable nucleotide-binding domain is a Streptococcus pyogenes Cas 9 (SpCas9) or a Staphylococcus aureus (SaCas9).

19. The system of any one of claims 10-18, wherein the system comprises one or more of the following:(i) the gRNA comprises a spacer sequence having at least 80% sequence identity toSEQ ID NO: 1 and the polynucleotide-programmable nucleotide-binding domain is VQR-BE3;(ii) the gRNA comprises a spacer sequence having at least 80% sequence identity toSEQ ID NO: 2 and the polynucleotide-programmable nucleotide-binding domain is selected from: ABEmax- VRQR, NG-ABEmax, ABEmax, xABEmax, xCas9(3.6)-ABE, and xCas9(3.7)-ABE;(iii) the gRNA comprises a spacer sequence having at least 80% sequence identity toSEQ ID NO: 3 and the polynucleotide-programmable nucleotide-binding domain is SpCas9;(iv) the gRNA comprises a spacer sequence having at least 80% sequence identity toSEQ ID NO: 5 and the polynucleotide-programmable nucleotide-binding domain is selected from: CP-CBEmax, NG-ABEmax, and CP-ABEmax;(v) the gRNA comprises a spacer sequence having at least 80% sequence identity toSEQ ID NO: 7 and the polynucleotide-programmable nucleotide-binding domain is selected from: VRQR, NG, xBE, xCas9(3.6)-ABE, xCas9(3.6)-CBE, xCas9(3.7)-ABE, and xCas9(3.7)-CBE;(vi) the gRNA comprises a spacer sequence having at least 80% sequence identity toSEQ ID NO: 8 and the polynucleotide-programmable nucleotide-binding domain is selected from BE4max, AncBE4max, ABE8e, and ABEmax;(vii) the gRNA comprises a spacer sequence having at least 80% sequence identity toSEQ ID NO: 9 and the polynucleotide-programmable nucleotide-binding domain is all non-canonical ABE / CBE editors; and(viii) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 10 and the polynucleotide-programmable nucleotide-binding domain is SaCas9 or SaCas9-KKH;(ix) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 12 and the polynucleotide-programmable nucleotide-binding domain is SpCas9;(x) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 13 and the polynucleotide-programmable nucleotide-binding domain is SpCas9;(xi) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 14 and the polynucleotide-programmable nucleotide-binding domain is SpCas9;(xii) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 15 and the polynucleotide-programmable nucleotide- binding domain is SaCas9 or SaCas9-KKH;(xiii) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 16 and the polynucleotide-programmable nucleotide- binding domain is SaCas9 or SaCas9-KKH;(xiv) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 17 and the polynucleotide-programmable nucleotide- binding domain is Nme2Cas9 -ABE8e or NmeCas9-ABE8e;(xv) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 18 and the polynucleotide-programmable nucleotide- binding domain is Nme2Cas9 -ABE8e or NmeCas9-ABE8e;(xvi) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 19 and the polynucleotide-programmable nucleotide- binding domain is SauriABESe;(xvii) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 20 and the polynucleotide-programmable nucleotide- binding domain is SauriABE8e;(xviii) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 244 and the polynucleotide-programmable nucleotide- binding domain is Nme2Cas9 -ABE8e or NmeCas9-ABE8e; and(xix) the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 245 and the polynucleotide-programmable nucleotide- binding domain is Nme2Cas9 -ABE8e or NmeCas9-ABE8e.

20. The system of claim 19, wherein the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 8 and the polynucleotide-programmable nucleotide-binding domain is selected from BE4max, AncBE4max, ABE8e, and ABEmax.

21. The system of claim 19, wherein the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 3 and the polynucleotide-programmable nucleotide-binding domain is SpCas9.

22. The system of claim 19, wherein the gRNA comprises a spacer sequence having at least 80% sequence identity to SEQ ID NO: 2 and the polynucleotide-programmable nucleotide-binding domain is selected from: ABEmax- VRQR, NG-ABEmax, ABEmax, xABEmax, xCas9(3.6)-ABE, and xCas9(3.7)-ABE.

23. The system of any one of claims 10-22, wherein the gRNA and the polynucleotide- programmable nucleotide-binding domain are in a ribonucleoprotein complex (RNP).

24. The system of any one of claims 10-22, wherein the gRNA is a PEgRNA and the polynucleotide-programmable nucleotide-binding domain is a prime editor polypeptide.

25. The system of claim 24, wherein the prime editor polypeptide comprises a nickase and a reverse transcriptase.

26. A recombinant adeno-associated virus (rAAV) comprising: a capsid protein having at least 80% sequence identity to a sequence selected from SEQ ID NO: 212-222, 252-255; and an AAV genome comprising: a polynucleotide sequence encoding a gRNA selected from any of the gRNAs of claims 1-9; and a polynucleotide sequence encoding a polynucleotide-programmable nucleotide- binding domain.

27. A lipid nanoparticle (LNP) comprising: a polynucleotide sequence encoding a gRNA selected from any of the gRNAs of claims 1-9; and a polynucleotide sequence encoding a polynucleotide-programmable nucleotide- binding domain.

28. A lipid nanoparticle (LNP) comprising: a ribonucleoprotein complex (RNP) comprising a gRNA selected from any of the gRNAs of claims 1-9 and a polynucleotide-programmable nucleotide-binding domain.

29. The rAAV or LNP of any one of claims 26-28, wherein the polynucleotide- programmable nucleotide-binding domain is selected from: a base editor, a prime editor, a Cas9, a Cas9 nickase, a dead Cas9, a Casl2a, a Casl2a nickase, a dead Casl2a, a FokI nuclease, an integrase, a zinc finger nuclease, or a transcription activator-like effector nuclease.

30. The rAAV or LNP of claim 29, wherein the polynucleotide-programmable nucleotide- binding domain is a base editor.

31. The rAAV or LNP of claim 30, wherein the base editor is a cytosine base editor (CBEs).

32. The rAAV or LNP of claim 30, wherein the base editor is a adenine base editor (ABEs).

33. The rAAV or LNP of claim 30, wherein the base editor is a non-canonical CBE base editor or a non-canonical ABE base editor.

34. The rAAV or LNP of claim 30, wherein the base editor is selected from: ABEmax- VRQR, ABEmax-NG, ABEmax, xABEmax, xCas9(3.6)-ABE, xCas9(3.7)-ABE, CP- CBEmax, CP-ABEmax, BE3-xCas9-3.6, BE3-xCas9-3.7, BE4max-xCas9(3.6), BE4max- xCas9(3.7)-, BE4max, BE4max-CP1028, BE3-pBK-VQR, AncBE4max, CBE6a, CBE6b, CBE6c, CBE6d, ABE8e, ABE8e-NG, ABE8e-CP1028, pCMV-SpRY-ABE8e, ABE8.20- d, ABE8.20-m, ABE8.17-m, ABE8e-CP1041, ABEmax-CP1012, ABE7.10-3.6, ABE7.10-3.7, ABE7.10, and pCAG-CBE4max-SpRY.

35. The rAAV or LNP of claim 29, wherein the polynucleotide-programmable nucleotide- binding domain is Streptococcus pyogenes Cas 9 (SpCas9) or Staphylococcus aureus (SaCas9).

36. An engineered cell comprising: a genetic modification in the 3’ UTR of PKD1, wherein the engineered cell exhibits increased PKD 1 mRNA levels as compared to a cell that does not have a genetic modification in the 3’ UTR of PKD1.

37. The engineered cell of claim 36, comprising: a guide RNA (gRNA) comprising: a spacer sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 1-10, 12-20, 244, 245; and a scaffold capable of associating with a polynucleotide-programmable nucleotide- binding domain and guiding the polynucleotide-programmable nucleotide-binding domain to a sequence complementary to the spacer sequence.

38. The engineered cell of claim 37, comprising: a base editor, wherein the base editor is selected from: ABEmax-VRQR, ABEmax-NG, ABEmax, xABEmax, xCas9(3.6)-ABE, xCas9(3.7)-ABE, CP-CBEmax, CP-ABEmax, BE3-xCas9-3.6, BE3-xCas9-3.7, BE4max-xCas9(3.6), BE4max-xCas9(3.7)-, BE4max, BE4max- CP1028, BE3-pBK-VQR, AncBE4max, CBE6a, CBE6b, CBE6c, CBE6d, ABE8e, ABE8e-NG, ABE8e-CP1028, pCMV-SpRY-ABE8e, ABE8.20-d, ABE8.20-m, ABE8.17-m, ABE8e-CP1041, ABEmax-CP1012, ABE7.10-3.6, ABE7.10-3.7, ABE7.10, and pCAG-CBE4max-SpRY.

39. The engineered cell of any one of claims 36-38, wherein the cell comprises a kidney cell.

40. A method for increasing PKD1 mRNA levels in a cell, the method comprising: introducing into the cell a system of any one of claims 10-23, an rAAV of any one claims 26 and 29-35, an LNP of any one of claims 27-35, or a combination thereof, whereby, upon introducing the system into a cell, the system modifies a binding site of a miRNA-17 family miRNA in the 3’ UTR of PKD1, thereby preventing binding of the miRNA-17 family miRNA and de-repressing PKD1 mRNA levels.

41. The method of claim 40, wherein the cell is a kidney cell.

42. The method of claim 40 or 41, wherein the cell or kidney cell is in vivo.

43. A method of treating a subject having or is suspected of having a PKD-associated disorder, the method comprising: introducing into the cell a system of any one of claims 10-23, a rAAV of any one claims 26 and 29-35, a LNP of any one of claims 27-35, or a combination thereof, whereby, upon introducing the system into the subject, the system modifies a binding site of a miRNA-17 family miRNA in the 3’ UTR of PKD1, thereby preventing binding of the miRNA-17 family miRNA and de-repressing PKD1 mRNA levels.

44. The method of claim 43, wherein the PKD-associated disorder is autosomal dominant polycystic kidney disease (ADPKD).

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