Gene editing system targeting hydroxyacid oxidase 1 (HAO1) and uses thereof for treating primary hyperoxaluria 1 (PH1)

Abstract

A method for treating primary hyperoxaluria 1 (PH1), comprising administering to a subject in need thereof an effective amount of a gene editing composition, which comprises: (a) a messenger RNA (mRNA) encoding a Cas12i2 polypeptide, (b) a guide RNA (gRNA) comprising a spacer sequence, which is specific to a target sequence in the HAO1 gene; and (c) and lipid excipients, which comprise a cationic lipid, a zwitterionic phospholipid, cholesterol, a pegylated lipid, or a combination thereof. In some instances, the gene editing composition is administered to the subject an amount of total RNA of about 0.4 mg / kg to about 1 mg / kg.

Description

[0001] GENE EDITING SYSTEM TARGETING HYDROXYACID OXIDASE 1 (HAO1) AND USES THEREOF FOR TREATING PRIMARY HYPEROXALURIA 1 (PHI)

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This Patent Application claims priority from US Patent Application No. 63 / 759,748 filed on February 18, 2025 and US Patent Application No. 63 / 912,959 filed on November 6, 2025, the entire disclosure of which is incorporated herein by reference.

[0004] BACKGROUND OF THE INVENTION

[0005] Primary Hyperoxaluria (PH) is a medical condition characterized by recurrent kidney and bladder stones resulting from overproduction of oxalate, which combines with calcium to form calcium oxalate, a main component of kidney and bladder stones. PH is estimated to affect 1 out of 58,000 individuals worldwide. There are three types of PH, PHI, PH2, and PH3, among which PHI accounts for approximately 80% of the cases.

[0006] Primary Hyperoxaluria is associated with genetic mutations in genes such as AGXT, GRHPR, and HOGA1 genes, leading to abnormal breakdown of glyoxylate. Glycolate oxidase (GO), encoded by the hydroxyacid oxidase 1 (HAO1) gene, converts glyoxylate to oxalate. Knocking out the HAO1 gene could reduce the level of GO enzyme and, in turn, the level of oxalate, thereby benefiting PH treatment.

[0007] SUMMARY OF THE INVENTION

[0008] The present disclosure is based, at least in part, on the development of an HAO 1 -targeting gene editing composition, which successfully edited the HAO1 gene and reduced serum glycolate levels and GO enzyme activity in a non-human primate (NHP) animal model. Based on at least the NHP animal data, efficacious doses of the gene editing composition for treating PHI in human patients were determined by a quantitative systems pharmacology (QSP) model. Accordingly, provided herein are methods for treating PHI with the HAO 1 -targeting gene editing composition as also disclosed herein. Provided herein is also a gene editing composition as mentioned above for use in treating PHI in a subject.In some aspects, the present disclosure provides a method for treating primary hyperoxaluria 1 (PHI), the method comprising administering to a subject in need thereof an effective amount of a gene editing composition that targets the HA01 gene. In some embodiments, the HAO 1 -targeting gene editing composition comprises: (a) a messenger RNA (mRNA) encoding a Casl2i2 polypeptide, which comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1, the Casl2i2 polypeptide comprising at least amino acid substitutions D581R, I926R, and VI 03 OG relative to SEQ ID NO: 1; (b) a guide RNA (gRNA) comprising a spacer sequence, which is specific to a target sequence of 5’-CAAAGTCTATATATGACTAT-3’ (SEQ ID NO: 10) in the HAO1 gene; and (c) lipid excipients, which comprise a cationic lipid, a zwitterionic phospholipid, cholesterol, a pegylated lipid, or a combination thereof. In some embodiments, the mRNA and gRNA are associated with the lipid excipients. In some examples, the lipid excipients form lipid nanoparticles (LNPs) and the mRNA and gRNA are attached to or encapsulated by the LNPs.

[0009] In some embodiments, the gene editing composition is administered to the subject at an amount of total RNA of about 0.4 mg / kg to about 1 mg / kg. In one example, the amount of total RNA administered to the subject is about 0.4 mg / kg. In another example, the amount of total RNA administered to the subject is about 0.7 mg / kg. In yet another example, the amount of total RNA administered to the subject is or about 1.0 mg / kg. In some instances, the amount of total RNA administered to the subject does not exceed 75 mg, regardless of the subject’s body weight. In some embodiments, the gene editing composition is administered to the subject via intravenous infusion, for example, over 2 hours.

[0010] In some embodiments, the Casl2i2 polypeptide in the HAO 1 -targeting gene editing composition disclosed herein may comprise D581R, I926R, and V1030G relative to SEQ ID NO: 1. In other embodiments, the Casl2i2 polypeptide in the HAO 1 -targeting gene editing composition disclosed herein may comprise D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G relative to SEQ ID NO: 1. In one example, the Casl2i2 polypeptide comprises the amino acid sequence of SEQ ID NO: 2. In another example, the Casl2i2 polypeptide comprises the amino acid sequence of SEQ ID NO: 3.

[0011] In some embodiments, any of the Casl2i2 polypeptides may further comprise one or more functional domains, such as one or more nuclease localization signals (NLSs) and / or more peptide linkers. In some examples, the Casl2i2 polypeptide may further comprise a first nuclear localization signal (NLS) at the N-terminus, a second nuclear localization signal (NLS) at the C-terminus, or both, of the Casl2i2 polypeptide. In some instances, the first NLS and the second NLS are identical. In some specific examples, the first NLS and / or the second NLS is set forth as KRPAATKKAGQAKKKK (SEQ ID NO: 5). Alternatively, or in addition, the Casl2i2 polypeptide may further comprise a peptide linker between the NLS and the rest of the polypeptide. In some examples, the peptide linker is set forth as SAGGGGSGGGGSGGGGSG (SEQ ID NO: 6).

[0012] In some specific examples, the Casl2i2 polypeptide in the HAO 1 -targeting gene editing composition disclosed herein may comprise the first NLS at the N-terminus and the second NLS at the C-terminus, and the peptide linker connecting each of the first NLS and the second NLS to the rest of the polypeptide. In some instances, the first and second NLS both are set forth as SEQ ID NO: 5. Alternatively, or in addition, the peptide linker is set forth as SEQ ID NO: 6.

[0013] In one specific example, the Casl2i2 polypeptide comprises the amino acid sequence of SEQ ID NO: 7. In that case, the mRNA molecule in the HAO 1 -targeting composition disclosed herein may comprise a coding sequence at least 80% identical to SEQ ID NO: 8 and encodes the amino acid sequence of SEQ ID NO: 7.

[0014] In some embodiments, the mRNA molecule in the HAO 1 -targeting composition disclosed herein may comprise a 5’ cap moiety, a 3’ polyadenylation tail, or a combination thereof. Alternatively, or in addition, the mRNA may comprise one or more modified nucleotides, for example, pseudouridine residues in replacement of uridine residues. In some instances, at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or higher) of the uridine positions in the mRNA contain pseudouridines. In some specific examples, all of the uridine positions in the mRNA are pseudouridine residues. In some examples, the mRNA comprises the nucleotide sequence of SEQ ID NO: 23. Such an mRNA molecule may comprise a 5’ cap moiety, and pseudouridine residues at all uridine positions in the mRNA.

[0015] In some embodiments, the gRNA in the HAO 1 -targeting composition disclosed herein may comprise the spacer sequence set forth as 5’-CAAAGUCUAUAUAUGACUAU-3’ (SEQ ID NO: 11). The gRNA may further comprise a direct repeat sequence (e.g., 23-26 nucleotide in length). In some examples, the direct repeat sequence is 5’-AGAAAUCCGUCUUUCAUUGACGG-3’ (SEQ ID NO: 12). In some instances, the gRNA may comprise the nucleotide sequence of 5’-AGAAAUCCGUCUUUCAUUGACGGCAAAGUCUAUAUAUGACUAU-3’ (SEQ ID NO: 13). Any of the gRNAs provided herein may comprise one or more modifications, e.g.,phosphorothioate linkage and 2’-O-methylation. In some instances, the modifications may be located at the 5’ and / or 3’ nucleotides. In one specific example, the gRNA is a modified RNA set forth as SEQ ID NO: 14.

[0016] In some embodiments, the lipid excipients comprise in the HAO 1 -targeting composition disclosed herein may comprise: (a) between 40 and 50 mol% of a cationic lipid; (b) a neutral lipid, (c) a steroid; and (d) a polymer conjugated lipid. In some instances, the neutral lipid is DSPC, the steroid is cholesterol, and / or the polymer conjugated lipid is a pegylated lipid.

[0017] In some examples, the lipid excipients comprise between 45 to 48 mol% (e.g., between 47.2 to 47.8 mol%) of the cationic lipid. In some examples, the lipid excipients comprise between 5 and 15 mol% (e.g., between 9 and 11 mol%) of the neutral lipid. In some specific examples, the lipid excipients comprise about 10 mol%, of the neutral lipid. In some examples, the lipid excipients comprise between 32 and 40 mol% (e.g., between 39 to 49 mol%) of the steroid. In specific examples, the lipid excipients may comprise 40 mol% of the steroid.

[0018] In some embodiments, the subject for treatment by any of the methods disclosed herein is a human patient diagnosed with PHI . In some examples, the human patient carries a mutation in the AG T gene. In some examples, the human patient can be an adult patient (e.g., 18 to 64 years of age). In other examples, the human patient can be a pediatric patient (e.g., 6 to 17 years of age).

[0019] In some embodiments, the subject for treatment by any of the methods disclosed herein may have one or more of the following features: (a) preserved kidney function at eGFR > 30 mL / min / 1.73 m2; (b) mean 24-hour urinary oxalate > 0.7 mmol / 24 hours / 1.73 m2; and (c) no evidence of systemic oxalosis.

[0020] In some embodiments, the subject for treatment by any of the methods disclosed herein may receive treatment of an anti-inflammation agent (e.g., dexamethasone), an anti-histamine agent (e.g., Hi blocker or H2 blocker), or a combination thereof prior to the treatment, after the treatment, or a combination thereof.

[0021] In some examples, the subject is an adult patient and may receive dexamethasone (e.g., at 8 or 10 mg) 16-24 hours prior to the administration of the gene editing composition. Alternatively, or in addition, the adult patient may receive, 1-2 hours prior to the administration of the gene editing composition, (i) dexamethasone orally or by intravenous injection (e.g., at 10 mg), (ii) an Hi blocker by intravenous injection (e.g., diphenhydramine at 50 mg or an equivalent thereof), or Hi blocker orally (e.g., cetirizine at 10 mg or an equivalent thereof); (iii) an H2 blocker orally orby intravenous injection (e.g., famotidine at 20 mg or an equivalent thereof); and / or (iv) acetaminophen / paracetamol by intravenous injection (e.g. 15 mg / kg for <50 kg or 750 mg for >50 kg) . Alternatively, or in addition, the adult patient may receive, 6 hours after the administration of the gene editing composition, dexamethasone by intravenous injection (e.g., at 4 mg). Alternatively, or in addition, the adult patient may receive, 24 hours after the administration of the gene editing composition, dexamethasone by intravenous injection (e.g., at 4 mg).

[0022] In some examples, the subject is a pediatric patient and may receive, 16-24 hours prior to the administration of the gene editing composition, dexamethasone orally (e.g., at 0.15 mg / kg to 8 mg). Alternatively, or in addition, the pediatric patient and may receive, 1-2 hours prior to the administration of the gene editing composition, (i) dexamethasone by intravenous injection (e.g., at 0.18 mg / kg to 10 mg); (ii) an Hi blocker by intravenous injection (e.g., diphenhydramine at 2 mg / kg and up to 50 mg or an equivalent thereof), or an Hi blocker orally (e.g., cetirizine at 10 mg or an equivalent thereof); (iii) an H2 blocker orally or by intravenous injection (e.g., famotidine at 0.25 mg / kg and up to 20 mg or an equivalent thereof); and / or (iv) acetaminophen / paracetamol by intravenous injection (e.g. 15 mg / kg for <50 kg or 750 mg for>50 kg). Alternatively, or in addition, the pediatric patient and may receive, 6 hours after the administration of the gene editing composition, dexamethasone by intravenous injection (e.g., at 0.075 mg / kg and up to 4 mg). Alternatively, or in addition, 24 hours after the administration of the gene editing composition, dexamethasone by intravenous injection (e.g., at 0.075 mg / kg and up to 4 mg).

[0023] Any of the methods disclosed herein may further comprise measuring levels of GO enzyme, urinary oxalate, and / or serum glycolate after the administration of the gene editing composition. Alternatively, or in addition, the method may further comprise monitoring occurrence of an adverse event after the administration of the gene editing composition; and managing the adverse event when occurrence is observed.

[0024] Also within the scope of the present disclosure are any of the HAO 1 -targeting gene editing compositions for use in treating PHI in a subject in need thereof (e.g., a human adult patient or a human pediatric patient), as well as uses of the HAO 1 -targeting gene editing compositions for manufacturing a medicament for treatment of PHI.

[0025] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawing in combination with the detailed description of specific embodiments presented herein.

[0027] FIGs. 1A-1F include diagrams showing serum glycolate levels in non- human primates (NHPs) receiving a single dose of the HAO 1 -targeting gene editing composition via intravenous infusion (IV) at 2.25 mg / kg, 2.5 mg / kg, 3.0 mg / kg doses (total RNA amount in the gene editing composition). FIG. 1A: serum glycolate levels in treated male and female groups combined. FIG. IB: Serum glycolate levels in treated males. FIG. 1C: Serum glycolate levels in treated females.

[0028] FIG. ID: Fold of change of serum glycolate levels relative to pre-treatment serum glycolate levels in treated male and female groups combined. FIG. IE: Fold of change of serum glycolate levels relative to pre-treatment serum glycolate levels in treated females. FIG. IF: Fold of change of serum glycolate levels relative to pre-treatment serum glycolate levels in treated males.

[0029] FIGs. 2A and 2B include diagrams showing GO enzyme activity in all NHPs receiving a single dose of the HAO 1 -targeting gene editing composition via intravenous infusion (IV) at 2.25 mg / kg, 2.5 mg / kg, 3.0 mg / kg doses (total RNA amount in the gene editing composition). FIG.3A:

[0030] GO enzyme activity all HNPs (males and females). FIG. 3B: GO enzyme activities at each dose in male and female NHP groups.

[0031] FIG. 3 is a diagram showing the relationship between GO enzyme activity and HAO1 editing in the livers of non-human primates (NHPs) receiving a single dose of the HAO 1 -targeting gene editing composition via intravenous infusion (IV) at 2.25 mg / kg, 2.5 mg / kg, 3.0 mg / kg doses (total RNA amount in the gene editing composition).

[0032] DETAILED DESCRIPTION OF THE INVENTION

[0033] Primary hyperoxaluria (PH) is a group of rare genetic metabolic disorders characterized by oxalate accumulation in the kidneys and other organ systems (Milliner et al., GeneReviews®, 2022, Settle WA:University of Washington Press: 1993-2023). PH is inherited in an autosomal recessive pattern. There are 3 types of PH, which are differentiated by a specific enzyme deficiency (Cochat et al., N. Engl. J. Med., 2013, 369(7): 649-658) caused by a broad range of underlyingmutations to the respective genes. Expression of these different enzymes is found primarily in the liver in PHI and primary hyperoxaluria type 2 (PH2), and in the kidney and liver in primary hyperoxaluria type 3 (PH3) (Forbes et al., Br. J. Clin. Pharmacol., 2022, 88(6): 2525-2538).

[0034] PHI accounts for approximately 80% of PH cases and is the most clinically severe (Hoppe, Nat. Rev. Nephrol., 2012, 8(8): 467-475). PHI results from a mutation of the AGXT gene, which is specifically expressed in hepatocytes, leading to low alanine-glyoxylate aminotransferase (AGT) enzyme activity. Reduced or absent AGT enzyme activity leads to impaired metabolism of glyoxylate into glycine, with the excess substrate then converted into insoluble oxalate, leading to elevated plasma levels and a large increase in urinary oxalate excretion (UOx). Urinary supersaturation of calcium oxalate promotes renal stone formation, progressive nephrocalcinosis, and eventually renal failure (Hoppe, 2012). Even while renal impairment is still moderate, these symptoms cause substantial clinical burden and healthcare resource utilization in patients with PHI, such as hospitalizations, emergency room visits, and numerous stone management interventions, frequently with complications. Collectively, the current clinical and treatment burden highlights the need for an effective PHI treatment (Wang et al., Front Med., 2021, 8:703305).

[0035] The current standard of care includes hyperhydration, crystallization inhibitors, dietary oxalate restriction, pyridoxine (if the mutation is responsive to supplementation), renal replacement therapies when required, and small interfering RNA (siRNA) therapy when available (Oxlumo® [lumasiran] or Rivfloza™ [nedosiran]). Hyperhydration, which is a cornerstone of therapy, is burdensome, creating adherence issues that necessitate gastrostomy tube placement in some patients, and has a significant impact on quality of life (Sas et al., Front Med. (Lausanne), 2021, 8:592357; and Garrelfs et al., Kidney Int., 2023, 103(5):990-993). A small fraction (<5%) of patients are completely responsive to pyridoxine, while partial responders likely still have increased urinary oxalate and thus are at increased risk for end-stage renal disease (ESRD). The ability of siRNA therapies to lower urinary oxalate has provided encouraging early clinical results in patients with PHI, but barriers to access and monthly or quarterly subcutaneous treatments remain a challenge. Finally, liver transplantation (with or without a kidney transplant), while curative, is a surgical procedure with high morbidity and mortality, requiring long-term immunosuppressants and potential graft rejection, particularly in the PHI population. Additional safe and efficacious therapies that address these challenges and improve long-term outcomes areneeded. A one-time therapy that provides durable control of urinary oxalate could improve adherence, as well as reduce ESRD rates and limit disease progression to systemic oxalosis.

[0036] The present disclosure reports that the HAO 1 -targeting gene editing composition provided herein, at specific doses (total RNA amount in the composition), successfully reduced serum glycolate levels and GO enzyme activity in a non-human primate (NHP) animal model. It is further reported that the reduction of GO enzyme activity correlates the HA01 gene editing level. See Example 1 below. Accordingly, the present disclosure provides methods for treating PHI via genetic editing of the HA01 by the HAO 1 -targeting gene editing composition at specific doses, which are determined by standard allometric scaling based on the doses used in the NHP animal model. In view of the data from the NHP animal model (e.g., those reported herein), the PHI treatment method would be expected to be effective in alleviating PHI symptoms via, e.g, reducing GO enzyme activity via genetic editing of the HA01 gene, leading to reduction of serum glycolate, so as to benefit treatment of PHI in a patient.

[0037] I. Compositions for Genetic Editing of HAO1 Gene

[0038] Hydroxyacid oxidase 1 (“HA01”), also known as “glycolate oxidase 1,” is a peroxisome protein expressed primarily in the liver and pancreas, and its activities include oxidation of glycolate and 2-hydroxy fatty acids. The HA01 gene product is the glycolate oxidase (GO) enzyme, also referred to as the GO protein or HA01 protein. The structural information of human HA01 gene can be found under NCBI Reference Sequence NG 046733.1.

[0039] The present disclosure provides a gene editing composition targeting an HA01 gene, e.g, the human HA01 gene. The HA01 gene editing composition provided herein may comprise: (a) a Casl2i2 polypeptide or a nucleic acid encoding the Casl2i2 polypeptide, (b) a guide RNA targeting a genomic site in an HA01 gene, and (c) lipid excipients comprising one or more lipids, which may form lipid nanoparticle (LNP) structures. The Casl2i2 polypeptide or the encoding nucleic acid and / or the gRNA can be associated with the lipid excipients. When the lipid excipients form LNPs, the Casl2i2 polypeptide or the encoding nucleic acid and / or the gRNA may be attached to and / or encapsulated by the LNPs.

[0040] In some embodiments, the HA01 gene editing composition may comprise a messenger RNA (mRNA) molecule encoding the Casl2i2 polypeptide, which may comprise an amino acid sequence at least 95% identical to a parent Casl2i2 CRISPR nuclease set forth as SEQ ID NO: 1and comprise at least amino acid substitutions at positions D581, 1926, and V1030 (e.g., D581R, I926R, and V1030G) relative to SEQ ID NO: 1. Alternatively, or in addition, the gRNA in the HA01 gene editing composition may comprise a spacer sequence, which is specific to a target sequence of 5’-CAAAGTCTATATATGACTAT-3’ (SEQ ID NO: 10) in the HAO1 gene. The mRNA and the gRNA in the composition may have a ratio of l±20%, for example, l±10%, 1±5% or 1±2%. In some examples, the ratio is about 1 : 1 (e.g., 1). Further, both the mRNA and the gRNA can be associated with (e.g., attached to or encapsulated by) the lipid excipients, which may form LNPs.

[0041] A. Casl2i2 Polypeptides and Encoding Nucleic Acids

[0042] The HA01 gene editing composition provided herein involves a Casl2i2 polypeptide or a nucleic acid encoding the Casl2i2 polypeptide. In specific examples, the HA01 gene editing composition comprises a messenger RNA (mRNA) molecule encoding the Casl2i2 polypeptide.

[0043] (a) Casl2i2 Polypeptide

[0044] The Casl2i2 polypeptide disclosed herein comprises a Casl2i2 nuclease domain and optionally one or more additional functional motifs, for example, nuclear localization signal(s), peptide linkers, or a combination thereof. Casl2i2 nuclease is a Class 2 type V CRISPR-Cas endonuclease that is capable of cleaving target double-strand DNAs guided by guide RNAs. Casl2i2 nucleases can be found, e.g., in WO / 2021 / 202800, and WO2022 / 256642, the relevant disclosures of each of which are incorporated by reference for the subject matter and purpose referenced herein.

[0045] In some embodiments, the Casl2i2 polypeptide involved in the HA01 gene editing compositions disclosed herein may comprise a Casl2i2 nuclease, which is a variant of a parent Casl2i2 enzyme set forth in SEQ ID NO: 1. See the Sequence Table below. Such a variant Casl2i2 nuclease may comprise an amino acid sequence at least 90% (e.g., at least 95%, at least 97%, at least 98%, at least 99%, or above) identical to SEQ ID NO: 1. Homology or identity can be determined by amino acid sequence alignment, e.g, using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.

[0046] The “percent identity” of two nucleic acids or of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm isincorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength- 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0047] The variant Casl2i2 nuclease may comprise one or more amino acid substitutions relative to SEQ ID NO:1, for example, at positions D581, 1926, V1030, and optionally further at positions G624, F626, P868, E1035, and S1046 in SEQ ID NO: 1. In some instances, the original amino acid residue in SEQ ID NO: 1 at the just-noted positions (e.g., D581, G624, F626, 1926, and / or El 035 positions) may be replaced by R or a conservative substitution thereof (e.g., K). Alternatively, or in addition, the original amino acid residue in SEQ ID NO: 1 at the noted positions (e.g., VI 030 and / or SI 046) may be replaced with G or a conservative substitution thereof such as A. Further, the original amino acid residue at position P868 may be replaced with T or a conservative substitution thereof such as S.

[0048] As used herein, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds. , Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0049] In one example, the variant Casl2i2 nuclease comprises D581R, I926R, and V1030G relative to SEQ ID NO: 1. Such a Casl2i2 nuclease may comprise the amino acid sequence of SEQ ID NO: 2. See the Sequence Table below.In another example, the variant Casl2i2 nuclease comprises D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S 1046G relative to SEQ ID NO: 1. Such a Casl2i2 nuclease may comprise the amino acid sequence of SEQ ID NO: 3.

[0050] Any of the Casl2i2 polypeptides may further comprise one or more functional motifs in addition to the nuclease domain. For example, the Casl2i polypeptide comprises at least one (e.g., two, three, four, five, six, or more, identical or different) nuclear localization signal (NLS). In some embodiments, the Casl2i polypeptide comprises at least one (e.g., two, three, four, five, six, or more, identical or different) nuclear export signal (NES). In some embodiments, the Casl2i polypeptide comprises at least one (e.g., two, three, four, five, six, or more) NLS and at least one (e.g., two, three, four, five, six, or more) NES. In some examples, the Casl2i2 polypeptide may comprise one NLS at the C-terminus. In other examples, the Casl2i2 polypeptide may comprise one NLS at the N-terminus and one NLS at the C-terminus. In some instances, the N-terminus and C-terminus NLS motifs can be identical. Suitable NLS motifs known in the art can be used in the Casl2i2 polypeptides disclosed herein. An example is provided in the Sequence Table below.

[0051] In some embodiments, the Casl2i2 polypeptide may comprise one or more flexible peptide linkers between the nuclease domain and the one or more additional functional motifs such as the NLS motifs. Any suitable flexible peptide linkers (e.g., a G / S rich peptide linker as those known in the art) can be used. One example is provided in the Sequence Table below.

[0052] In one specific example, the Casl2i2 polypeptide may comprise, from N-terminus to C-terminus, a Casl2i2 nuclease domain and an NLS. In another specific example, the Casl2i2 polypeptide may comprise, from N-terminus to C-terminus, a first NLS, a first flexible peptide linker, a Casl2i2 nuclease domain, a second flexible peptide linker, and a second NLS. Such a Casl2i2 polypeptide may comprise the amino acid sequence of SEQ ID NO: 7. In another example, the Casl2i2 polypeptide may comprise the amino acid sequence of SEQ ID NO: 17. See Sequence Table below. The NLS motif is italicized and in boldface and the peptide linker is underlined.

[0053] (b) Casl2i2-Encoding mRNA

[0054] In some embodiments, the H0A1 gene editing composition disclosed herein may comprise a nucleic acid such as an mRNA molecule that encodes the Casl2i2 polypeptide disclosed herein.

[0055] In some examples, the nucleotide sequence encoding the Casl2i polypeptide described herein can be codon-optimized for use in a particular host cell or organism, e.g., in human livercells. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp / codon / and these tables can be adapted in a number of ways. See Nakamura et al. Nucl. Acids Res. 28:292 (2000), which is incorporated herein by reference for the subject matter and purpose referenced herein. Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA). In some examples, the nucleic acid encoding the Casl2i polypeptides such as Casl2i2 polypeptides as disclosed herein can be an mRNA molecule, which can be codon optimized. Exemplary coding sequences, SEQ ID NO: 4 (encoding the Casl2i2 polypeptide of SEQ ID NO:7) and SEQ ID NO: 18 (encoding the Casl2i2 polypeptide of SEQ ID NO: 17), are provided in the Sequence Table below.

[0056] In addition to the coding sequence for the Casl2i2 polypeptide, the nucleic acid such as the mRNA molecule may further comprise an untranslated region (UTR) at the 5’ end and / or the 3’ end. For example, the mRNA molecule may comprise 5’ cap modification. mRNAs typically contain a 5 ’-cap for efficient translation, stabilization, and transportation in eukaryotic cells. In some examples, the mRNA molecule disclosed herein may contain the native 5’ m7G cap. Alternatively, the mRNA molecule may contain a non-naturally occurring, synthetic 5’ cap such as those known in the art for use in mRNAs, e.g., the synthetic caps provided by TriLink BioTechnologies, See also Ishikawa et al., Nucleic Acid Symposium Series No. 53, pages 129-130, the relevant disclosures of which are incorporated by reference for the purpose and subject matter referenced herein. Examples include, but are not limited to, CAP (G(5')ppp(5')G), mCAP (m7G(5')ppp(5')G), ARCA (3'-O-Me-m7G(5')ppp(5')G), m7(3’OMeG)(5’)ppp(‘5)m6(2’OMeA)pG, m7G(5')ppp(5')(2'OMeA)pU, and m7G(5')ppp(5')(2'OMeA)pG.

[0057] Alternatively, or in addition, the mRNA molecules disclosed herein may comprise one or more modifications, e.g., modified nucleotides and / or modified backbone linkages. In one example, the mRNA molecule may comprise pseudouridine residues. In some instances, the content of pseudouridine residues in the mRNA may be at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, or higher) at the uridine positions in the mRNA. In some examples, the mRNA may comprise pseudouridine residues at all uridine positions in the mRNA.

[0058] In one specific example, the mRNA in the HA01 gene editing composition disclosed herein may comprise a nucleotide sequence at least 70% identical to SEQ ID NO: 8 and encode aCasl2i2 polypeptide (e.g., comprising the amino acid sequence of SEQ ID NO: 7). For example, the mRNA may comprise a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or above identical to SEQ ID NO: 8 and encodes the Casl2i2 polypeptide of SEQ ID NO: 7. In one specific example, the mRNA comprises (e.g., consists of) the nucleotide sequence of SEQ ID NO: 8.

[0059] In another specific example, the mRNA in the HA01 gene editing composition disclosed herein may comprise a nucleotide sequence at least 70% identical to SEQ ID NO: 18 and encode a Casl2i2 polypeptide (e.g., comprising the amino acid sequence ofSEQ ID NO: 17). For example, the mRNA may comprise a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or above identical to SEQ ID NO: 18 and encodes the Casl2i2 polypeptide of SEQ ID NO: 17. In one specific example, the mRNA comprises (e.g., consists of) the nucleotide sequence of SEQ ID NO: 18.

[0060] Any of such mRNA molecules may contain a 5’ cap, e.g, the m7G(5')ppp(5')(2'OMeA)pG cap, and at least 75% (e.g, at least 80%, at least 85%, at least 90%, at least 95% pseudouridine residues at uridine positions in the mRNA. In one example, the mRNA contains pseudouridine residues at all uridine positions.

[0061] Additional modifications to RNA molecules as known in the art (e.g, provided herein) may be introduced into the Casl2i2-encoding mRNA molecules disclosed herein.

[0062] Alternatively, or in addition, the Casl2i2 polypeptide-encoding mRNA may contain a 3’ poly A tail. In some instances, the poly A tail in the mRNA may contain about 50-150 adenosine (A) residues. In some examples, the poly A tail may have a length of about 60-140 A residues. In other examples, the poly A tail may have a length of about 70-140 A residues or about 80-140 A residues. In some examples, the poly A tail may have a length of about 85-135 A residues. In one specific example, the Casl2i2 polypeptide-encoding mRNA may comprise the nucleotide sequence of SEQ ID NO: 23.

[0063] In some instances, the HAO 1 -targeting gene editing composition may comprise a population of mRNA molecules as disclosed herein with poly A tails of different lengths. In some examples, a substantial portion of the mRNA molecules (e.g, at least 70%) have poly A tails with lengths ranging from about 50 A residues to about 130 A residues (e.g, about 60-130 A residues or about 70-120 A residues). In some instances, the mRNA molecules may have an average poly A length of 70 A residues or greater, for example, > 80 A residues, > 85 A residues, or > 90 Aresidues. In specific examples, the mRNA molecules may have an average poly A length ranging from 80-120 A residues. In other specific examples, the mRNA molecules may have an average poly A length ranging from 90-110 A residues or ranging from 90-100 A residues.

[0064] B. RNA Guides

[0065] The HAO1 gene editing composition disclosed herein also involves a guide RNA (gRNA) that targets the HAO1 gene or a nucleic acid encoding such. As used herein, the term “RNA guide” or “RNA guide sequence” refers to any RNA molecule (e.g., a modified RNA molecule) that facilitates the targeting of a CRISPR nuclease such as any of the Casl2i2 polypeptides described herein to a target sequence (e.g., a sequence of an HAO1 gene). For example, an RNA guide can be a molecule targeting a nucleic acid sequence (target sequence) in a target gene (e.g., an HAO1 gene in the present disclosure). More specifically, the RNA guide comprises a domain (spacer sequence) that is specific to the target sequence (i.e., being the RNA counterpart of the target sequence) in the target gene. In addition to the spacer sequence, an RNA guide may further comprise a direct repeat (DR) sequence, which is recognizable by the CRISPR nuclease. In some instances, the RNA guide can be a modified RNA molecule comprising one or more deoxyribonucleotides, for example, in the spacer sequence, which binds a sequence complementary to the target sequence. In some examples, the spacer sequence may contain a DNA sequence or a DNA / RNA hybrid sequence.

[0066] As used herein, the term “complementary” refers to a first polynucleotide (e.g., a spacer sequence of an RNA guide) that has a certain level of complementarity to a second polynucleotide (e.g., the complementary sequence of a target sequence) such that the first and second polynucleotides can form a double-stranded complex via base-pairing to permit an effector polypeptide that is complexed with the first polynucleotide to act on (e.g., cleave) the second polynucleotide. In some embodiments, the first polynucleotide may be substantially complementary to the second polynucleotide, i.e., having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementarity to the second polynucleotide. In some embodiments, the first polynucleotide is completely complementary to the second polynucleotide, i.e., having 100% complementarity to the second polynucleotide.

[0067] In some embodiments, the RNA guide may comprise a spacer sequence followed by adirect repeat sequence, referring to the sequences in the 5’ to 3’ direction. In some embodiments, the RNA guide may comprise a first direct repeat sequence followed by a spacer sequence and a second direct repeat sequence, referring to the sequences in the 5’ to 3’ direction. In some embodiments, the first and second direct repeats of such an RNA guide can be identical. In some embodiments, the first and second direct repeats of such an RNA guide may be different.

[0068] In some embodiments, the spacer sequence and the direct repeat sequence(s) of the RNA guide are present within the same RNA molecule. In some embodiments, the spacer and direct repeat sequences are linked directly to one another. In some embodiments, a short linker is present between the spacer and direct repeat sequences, e.g., an RNA linker of 1, 2, or 3 nucleotides in length. In some embodiments, the spacer sequence and the direct repeat sequence(s) of the RNA guide are present in separate molecules, which are joined to one another by base pairing interactions.

[0069] (a) Spacer Sequence

[0070] In some embodiments, the RNA guide in the HA01 gene editing composition provided herein comprises a spacer sequence specific to a target sequence in exon 1 of the human HA01 gene, the nucleotide sequence of which is provided in the Sequence Table below. The target sequence is adjacent to a 5’-NTTN-3’ or 5’-TTN-3’ “protospacer adjacent motif’ or “PAM” sequence, in which N represents any nucleotide sequence. In some instances, the target sequence is downstream to the PAM sequence, for example, at the 3’ end of the PAM sequence.

[0071] A PAM sequence is a DNA sequence adjacent to a target sequence, to which a complex comprising an RNA guide (e.g., an HAO 1 -targeting RNA guide) and a Casl2i2 polypeptide provided herein binds. In a double-stranded DNA molecule, the strand containing the PAM motif is called the “PAM-strand” and the complementary strand is called the “non-PAM strand.” The spacer sequence in an RNA guide can bind to a site in the non-PAM strand that is complementary to a target sequence disclosed herein.

[0072] In some embodiments, the PAM strand is a coding (e.g., sense) strand. In other embodiments, the PAM strand is a non-coding (e.g., antisense strand). Since an RNA guide binds the non-PAM strand via base-pairing, the non-PAM strand is also known as the target strand, while the PAM strand is also known as the non-target strand.

[0073] The spacer sequence of the RNA guide may have a length of between 15-30 nucleotides(e.g, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and is complementary to a non-PAM strand sequence. In some embodiments, the spacer sequence is designed to be complementary to a specific DNA strand, e.g., of a genomic locus, which may be within exon 1 of the human HAO1 gene. In specific examples, the target sequence is set forth as 5’-CAAAGTCTATATATGACTAT-3’ (SEQ ID NO: 10) in the HAO1 gene.

[0074] In some embodiments, the spacer sequence in the RNA guide is substantially identical to a complementary strand of a target sequence (e.g., SEQ ID NO: 10). In some embodiments, the spacer sequence may have at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to a complementary strand of a target sequence such as SEQ ID NO: 10. The percent identity between two such nucleic acids can be determined manually by inspection of the two optimally aligned nucleic acid sequences or by using software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters.

[0075] In some examples, the spacer sequence in the RNA guide disclosed herein may comprise the nucleotide sequence of 5’-CAAAGUCUAUAUAUGACUAU-3’ (SEQ ID NO: 11). Alternatively, the spacer sequence may comprise a nucleotide sequence at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or above identical to SEQ ID NO: 11.

[0076] In some embodiments, a spacer sequence described herein comprises an uracil (U). In some embodiments, a spacer sequence described herein comprises a thymine (T). Any nucleotide sequences provided here, unless explicitly noted, refers to a nucleic acid having the defined nucleotide sequence, regardless of modifications, if any, contained therein.

[0077] (b) Direct Repeat

[0078] Any of the RNA guides disclosed herein may further comprise a direct repeat sequence. In some embodiments, the direct repeat sequence of the RNA guide has a length of 15-30 nucleotides. In some examples, the direct repeat sequence may have a length of 20-30 nucleotides, for example, 23-26 nucleotides.

[0079] In some embodiments, the direct repeat sequence may comprise the nucleotide sequenceof 5’-AGAAAUCCGUCUUUCAUUGACGG-3’ (SEQ ID NO: 12). Alternatively, the spacer sequence may comprise a nucleotide sequence at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or above identical to SEQ ID NO: 12.

[0080] (c) Modifications

[0081] The RNA guide (and the mRNAs where applicable) provided herein may include one or more covalent modifications with respect to a reference sequence, in particular the parent polyribonucleotide, which are included within the scope of the present disclosure.

[0082] Exemplary modifications can include any modification to the sugar, the nucleobase, the internucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage / to the phosphodiester backbone), and any combination thereof. Some of the exemplary modifications provided herein are described in detail below.

[0083] The RNA guide may include any useful modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate / to a phosphodi ester linkage / to the phosphodiester backbone). One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the internucleoside linkage. Modifications may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additional modifications are described herein.

[0084] In some embodiments, the modification may include a chemical or cellular induced modification. For example, some nonlimiting examples of intracellular RNA modifications are described by Lewis and Pan in “RNA modifications and structures cooperate to RNA guide-protein interactions” from Nat Reviews Mol Cell Biol, 2017, 18:202-210.

[0085] Different sugar modifications, nucleotide modifications, and / or internucleoside linkages (e.g., backbone structures) may exist at various positions in the sequence. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of the sequence, such that the function of the sequence is not substantially decreased. The sequence may include from about 1% to about 100% modified nucleotides (either in relationto overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%>, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%).

[0086] In some embodiments, sugar modifications (e.g., at the 2’ position or 4’ position) or replacement of the sugar at one or more ribonucleotides of the sequence may, as well as backbone modifications, include modification or replacement of the phosphodiester linkages. Specific examples of a sequence include, but are not limited to, sequences including modified backbones or no natural internucleoside linkages such as internucleoside modifications, including modification or replacement of the phosphodiester linkages. Sequences having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this application, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, a sequence will include ribonucleotides with a phosphorus atom in its internucleoside backbone.

[0087] Modified sequence backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3 ’-alkylene phosphonates and chiral phosphonates, phosphinates, phosphorami dates such as 3 ’-amino phosphorami date and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3’-5’ linkages, 2’-5’ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3 ’-5’ to 5 ’-3’ or 2’ -5’ to 5 ’-2’. Various salts, mixed salts and free acid forms are also included. In some embodiments, the sequence may be negatively or positively charged.

[0088] The modified nucleotides, which may be incorporated into the sequence, can be modifiedon the internucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably. Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).

[0089] The a-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment.

[0090] In specific embodiments, a modified nucleoside includes an alpha -thio-nucleoside (e.g., 5’-O-(l-thiophosphate)-adenosine, 5’-O-(l-thiophosphate)-cytidine (a-thio-cytidine), 5’-O-(l-thiophosphate)-guanosine, 5’-O-(l-thiophosphate)-uridine, or 5’ -O-(l -thiophosphate)-pseudouridine).

[0091] Other internucleoside linkages that may be employed according to the present disclosure, including internucleoside linkages which do not contain a phosphorous atom, are described herein.

[0092] In some embodiments, the sequence may include one or more cytotoxic nucleosides. For example, cytotoxic nucleosides may be incorporated into sequence, such as bifunctional modification. Cytotoxic nucleoside may include, but are not limited to, adenosine arabinoside, 5-azacytidine, 4’-thio-aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, 1 -(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine, decitabine, 5-fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5-fluoro-l -(tetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione), troxacitabine, tezacitabine, 2’ -deoxy-2’ -methylidenecytidine (DMDC), and 6-mercaptopurine. Additional examples include fludarabine phosphate, N4-behenoyl-l-beta-D-arabinofuranosylcytosine, N4-octadecyl-l-beta-D-arabinofuranosylcytosine, N4-palmitoyl-l-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl) cytosine, and P-4055 (cytarabine 5 ’-elaidic acid ester).

[0093] In some embodiments, the sequence includes one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, poly -A sequence, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, etc). The one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of the more than one hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197) In some embodiments, the first isolated nucleic acid comprises messenger RNA (mRNA). In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of pyridin-4-one ribonucleoside, 5 -aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3 -methyluridine, 5-carboxymethyl-uridine, 1 -carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1 -taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1 -taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1 -methyl-pseudouridine, 4-thio-l-methyl-pseudouridine, 2 -thio- 1 -methyl-pseudouridine, 1 -methyl- 1 -deaza-pseudouri dine, 2-thio-1 -methyl- 1 -deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 5 -aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1 -methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza-pseudoisocytidine, 1 -methyl- 1 -deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-l -methyl-pseudoisocytidine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 2 -aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine. In some embodiments, mRNA comprises at least one nucleoside selected from the group consisting of inosine, 1 -methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1 -methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1 -methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0094] The sequence may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., naturally-occurring nucleotides, purine or pyrimidine, or any one or more or all of A, G, U, C, I, pU) may or may not be uniformly modified in the sequence, or in a given predetermined sequence region thereof. In some embodiments, the sequence includes a pseudouridine. In some embodiments, the sequence includes an inosine, which may aid in the immune system characterizing the sequence as endogenous versus viral RNAs. The incorporation of inosine may also mediate improved RNA stability / reduced degradation. See for example, Yu, Z. et al. (2015) RNA editing by AD ARI marks dsRNA as “self’. Cell Res. 25, 1283-1284, which is incorporated by reference in its entirety.

[0095] In some embodiments, one or more of the nucleotides of an RNA guide comprises a 2’ -O-methyl phosphorothioate modification. In some embodiments, each of the first three nucleotides of the RNA guide comprises a 2’-O-methyl phosphorothioate modification. In some embodiments, each of the last four nucleotides of the RNA guide comprises a 2’-O-methyl phosphorothioate modification. In some embodiments, each of the first to last, second to last, and third to last nucleotides of the RNA guide comprises a 2’-O-methyl phosphorothioate modification, and wherein the last nucleotide of the RNA guide is unmodified. In some embodiments, each of the first three nucleotides of the RNA guide comprises a 2’-O-methyl phosphorothioate modification, and each of the first to last, second to last, and third to last nucleotides of the RNA guide comprises a 2’-O-methyl phosphorothioate modification.When a gene editing system disclosed herein comprises nucleic acids encoding the Casl2i polypeptide disclosed herein, e.g., mRNA molecules, such nucleic acid molecules may contain any of the modifications disclosed herein, where applicable.

[0096] (d) Exemplary RNA Guides

[0097] In some examples, the RNA guide provided herein may comprise the direct repeat of SEQ ID NO: 12 and a spacer sequence having a length of 20 nucleotides, which is specific to a target sequence (e.g., SEQ ID NO: 10) within exon 1 of the HA01 gene. In some specific examples, the spacer sequence may consist of the nucleotide sequence of SEQ ID NO: 11. In some instances, the RNA guide may comprise, from 5’ to 3’, the direct repeat sequence and the spacer sequence.

[0098] In some specific examples, the RNA guide may comprise (e.g., consists of) the nucleotide sequence of SEQ ID NO: 13 provided in the Sequence Table below. Alternatively, the RNA guide may be a variant of SEQ ID NO: 13 comprising (e.g., consisting of) a nucleotide sequence at least 80% identical to SEQ ID NO: 13, for example, at least 85%, at least 90%, at least 95%, at least 97% or above identical to SEQ ID NO: 13. In some examples, the variant of SEQ ID NO: 13 may contain up to 5 nucleotides variations relative to SEQ ID NO: 13, for example, up to 4, 3, 2, or 1 nucleotide variations relative to SEQ ID NO: 13.

[0099] The RNA guide provided herein may comprise one or more modifications such as those disclosed herein. In some embodiments, the modifications comprise 2’-O-methylation and / or phosphothioate linkage. In some instances, such modifications can be located at the 5’ end and / or 3’ end of the RNA guide. One specific example is SEQ ID NO: 14 provided in the Sequence Table below.

[0100] C. Lipid Excipients and Lipid Nanoparticles (LNPs)

[0101] In some embodiments, the HAO1 gene editing compositions provided herein may comprise lipid excipients, which may comprise one or more types of lipids, e.g, a cationic lipid, a phospholipid such as a zwitterionic phospholipid, a pegylated lipid, cholesterol, or a combination thereof. The nucleic acid components (mRNA and RNA guide) can be associated with the lipid excipients.

[0102] The lipid excipients may form lipid nanoparticles (“LNPs”), which are particles comprising one or more lipids. In some embodiments, the lipid nanoparticles may comprise a monolayer lipid membrane. Examples of such LNPs include micelles and reverse micelles. In other embodiments,the LNPs may comprise a bilayer lipid membrane (e.g., liposomes). In yet other embodiments, the LNPs are multilamellar vesicles, which contain multiple lamellar phase lipid bilayers. Still in other embodiments, the LNP may be solid lipid nanoparticle, which comprises a solid lipid core matrix that can solubilize lipophilic molecules. In some instances, a solid lipid nanoparticle can also be used to solubilize molecules such as nucleic acid, which may be encapsulated based on charges. In a solid lipid nanoparticle, the lipid core can be stabilized by surfactants (emulsifiers) and cargos can be distributed into the lipid core.

[0103] The LNPs disclosed herein refer to particles, which may have at least one dimension on the order of nanometers (e.g. , 1-1 ,000 nm). The LNPs may contain a number of lipids, for example, cationic lipids, ionizable lipids, anionic lipids, neutral lipids, amphipathic lipids, conjugated lipids (e.g., PEGylated lipids), amphipathic lipids such as phospholipids, and / or cholesterol. In some embodiments, the LNPs provided herein are included in the gene editing composition and used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, tumor, and the like). In some embodiments, the active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), may be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g, an adverse immune response.

[0104] In some embodiments, the LNPs provided herein may comprise a cationic lipid (e.g., between 40 and 50 mol%), a neutral lipid (zwitterionic lipid) such as DSPC (e.g, between 5 and 15 mol%), a steroid such as cholesterol (e.g, between 32 and 40 mol%), and polymer conjugated lipid such as a pegylated lipid. See, e.g, WO2018081480, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein.

[0105] In some instances, the cationic lipid may constitute about 40-50 mol% in the LNPs of the gene editing composition disclosed herein. In some examples, the cationic lipid may constitute about 45 to 48 mol%. In other examples, the cationic lipid may constitute about 47.2 to 47.8 mol%.

[0106] In some instances, the neutral lipid may constitute about 5-15 mol% of the LNPs in the gene editing composition disclosed herein. In some examples, the neutral lipid may constitute about 9-11 mol%. In one example, the neutral lipid may constitute about 10 mol%.

[0107] In some instances, the LNPs may comprise the steroid such as cholesterol at about 32-40 mol%, for example, about 32-40 mol% or about 39-49 mol%. In one example, the LNPs maycomprise the steroid such as cholesterol at about 40 mol%.

[0108] (a) Cationic Lipids

[0109] In some embodiments, the lipid excipients comprise one or more cationic lipids, which are positively charged lipids.

[0110] A “cationic lipid” refers to a lipid capable of being positively charged. Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. Exemplary cationic lipids are ionizable such that they can exist in a positively charged or neutral form depending on pH. The ionization of the cationic lipid affects the surface charge of the lipid nanoparticle under different pH conditions. This charge state can influence plasma protein absorption, blood clearance and tissue distribution (Semple, S.C., et al., Adv. Drug Deliv Rev 32:3-17 (1998)) as well as the ability to form endosomolytic non-bilayer structures (Hafez, I.M., et al., Gene Ther 8: 1188-1196 (2001)) critical to the intracellular delivery of nucleic acids.

[0111] In some examples, the cationic lipid may have a positively charged hydrophilic head (amino head group, including an alkylamino or dialkylamino group) and a hydrophobic tail (e.g., one or two fatty acid or fatty alkyl chains) that are connected via a linker structure. In addition to these, the cationic lipid may also be a lipid including a cyclic amine group.

[0112] In one specific example, the cationic lipid is bis(2-butyloctyl) 10-(N-(3-(pyrrolidin-l-yl)propyl)nonanamido)nonadecanedioate (CL- A), the structure of which is provided below:

[0113]

[0114] a pharmaceutically acceptable salt or stereoisomer thereof.

[0115] Other exemplary cationic lipids include, but are not limited to, 3-(didodecylamino)-N 1 ,N 1 ,4-tridodecyl-l-piperazineethanamine (KL 10) , N 1 -[2-(didodecylamino)ethyl] -N 1 ,N4,N4-tridodecyl- 1 ,4-piperazinediethanamine (KL22), 14,25 -ditridecyl- 15 , 18 ,21 ,24-tetraaza-octatriacontane (KL25), l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2.2-dilinoleyl-4-dimethylaminomethyl-[l ,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31 -tetraen- 19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2.2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), 2-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l -amine (Octyl-CLinDMA), (2R)-2-({8- [(3P)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l-amine (Octyl-CLinDMA (2R)), (2S)-2-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy] propan- 1 -amine (Octyl-CLinDMA (2S)); 3-b-(N- (N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol); l,2-dioleoyl-3 -dimethylammonium propane (“DODAP”), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA); 1 ,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA); N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-l -aminium (DOBAQ); YSK05; 4-(((2,3-bis(oleoyloxy)propyl)(methyl)amino)methyl)benzoic acid (DOBAT); 3-((2,3-bis(oleoyloxy)propyl)(methyl)amino)propanoic acid (DOPAT); and Alny-100.

[0116] In other examples, the cationic lipids provided herein may be positively charged at a wide range of pH (e.g., pH of 1-12). Such cationic lipids may be amino lipids. In some instances, the cationic lipids may be pH-insensitive with a permanent positive charge. Examples of such cationic lipids include, but are not limited to, N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 1 ,2-Dioleyloxy-3 -trimethylaminopropane chloride salt (DOTAP.Q); N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), dioctadecylamidoglycyl carboxyspermine (DOGS); DODAC; N-(2,3-dioleyloxy)propyl-N,N- N-triethylammonium chloride (DOTMA); N-(l-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl-ammonium trifluoracetate (DOSPA); N-(2-carboxypropyl)-N,N-dimethyl-2, 3 -bis(oleoyloxy)propan-l -aminium (DOMPAQ); N- (carboxymethyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-l -aminium (DOAAQ); 0,0'-ditetradecanoyl-N-(alpha-trimethyl ammonio acetyl) diethanolamine chloride (DC-6-14); (1,2-dioleyloxypropyl)-3 dimethylhydroxyethyl ammoniumbromide) (DORIE), DODMA-An, N,N-distearyl-N,N-dimethylammonium chlorideHEPES, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (DSDAC), and N,N-distearyl-N,N-dimethylammonium bromide (DDAB). In some embodiments, the lipid nanoparticle comprises a combination of non-ionizable cationic lipids described above.

[0117] In some examples, the cationic lipid is an amino lipid. In some instances, the cationic lipid is the only amino lipid in the lipid excipients contained in the HA01 gene editing composition.(b) Amphipathic Lipids

[0118] In some embodiments, the lipid excipients disclosed herein comprises one or more amphipathic lipid, i.e., a lipid having a polar part and a non-polar part. Exemplary amphipathic lipids suitable for use in nanoparticles of the disclosure include, but are not limited to, sphingolipids, phospholipids, fatty acids, and amino lipids. In some examples, the lipid excipients comprise one or more phospholipids, for example, one or more saturated or (poly)unsaturated phospholipids or a combination thereof. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.

[0119] Exemplary phospholipids include, but are not limited to, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline. The fatty acid moieties in the phospholipids include, but are not limited to, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0120] Additional exemplary phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids.

[0121] In some examples, the phospholipids in the lipid excipients may be zwitterionic phospholipids. Examples include, but are not limited to, diacylphosphatidylcholine (or 1,2-Distearoyl-sn-glycero-3 -phosphocholine (DSPC)), diacylphosphatidylethanolamine, ceramide, cephalin, sterols (e.g., cholesterol) and cerebrosides. Other non-limiting examples of neutral lipids include dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), Dipalmitoylphosphatidylcholine (DOPG), 1 ,2-Dipalmitoyl-sn-glycero-3 -phosphoglycerol (DPPG), l,2-Dioleoyl-sn-glycerol-3 -phosphoethanolamine (DOPE), l-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1 ,2-Dipalmitoyl-sn-glycero-3 -phosphoethanolamine (DPPE), 1,2-Dimyristoyl-sn-glycero-3 -phosphoethanolamine (DMPE), 1 ,2-Distearoyl-sn-glycero-3 -phosphoethanolamine (DSPE), 16-0-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, l-stearoyl-2-oleoyl-phosphatidy ethanolamine (SOPE), and 1 ,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE), dipalmitoylphosphatidylcholine (DMPC), milk sphingomyelin, and 1,2-dilauroyl-sn-glycero-3 -phosphocholine (DLPC).

[0122] In one specific example, the lipid excipients in the HA01 gene editing composition provided herein comprise a zwitterionic phospholipid, which can be DSPC.

[0123] (c) PEGylated Lipids

[0124] In some embodiments, the lipid excipients disclosed herein comprise one or more PEGylated lipid. A PEGylated lipid (also known as a PEG lipid or a PEG-modified lipid) is a lipid modified with polyethylene glycol. A PEGylated lipid may be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols. For example, a PEGylated lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSG, or a PEG-DSPE lipid.

[0125] Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG chain. In certain embodiments, the PEG lipid is a PEG-OH lipid. As generally defined herein, a “PEG-OH lipid” (also referred to herein as “hydroxy -PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl (-OH) groups on the lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodiments, a PEG-OH or hydroxy-PEGylated lipid comprises an -OH group at the terminus of the PEG chain. In some examples, the PEG lipids may be modified to comprise a methoxy group (methoxy PEG or mPEG), which is a functional group consisting of a methyl moiety bound to oxygen.

[0126] Each possibility represents a separate embodiment of the present invention. In some embodiments, the length of the PEG chain comprises about 250, about 500, about 1000, about 2000, about 3000, about 5000, about 10000 ethylene oxide units.

[0127] In some examples, the lipid excipients in the HAO1 gene editing composition provided herein comprise a PEG lipid, which is 2-[2-(co-methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide (PL- A), the structure of which is provided below:

[0128]

[0129] inclusive)

[0130] Additional examples of pegylated lipids can be found in W02021 / 030701, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein.

[0131] (d) Exemplary LNPs

[0132] In some examples, the LNPs may comprise (e.g., consists of) a cationic lipid (e.g., bis(2-butyloctyl) 10-(N-(3-(pyrrolidin-l-yl)propyl)nonanamido)nonadecanedioate, a.k.a., CL-A), a zwitterionicphospholipid (e.g., DSPC), a pegylated lipid (e.g., 2-[2-(co-methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide; a.k.a., PL- A), and cholesterol. In certain examples, the molar ratio of the cationic lipid to the phospholipid lipid ranges from about 2:1 to about 8:1, the molar ratio of the cationic lipid to cholesterol ranges from about 2:1 to 1:1 and / or the molar ratio of the cationic lipid to the pegylated lipid ranges from about 100: 1 to about 25:1.

[0133] In some embodiments, the lipid excipients in the HA01 gene editing composition may comprise about 30-60 mol% of the cationic lipid, e.g, about 40-50 mol% of the cationic lipid, based on total mol of the lipid excipients in the LNP. In some examples, the lipid excipients in the gene editing composition may contain about 30 mol% of the cationic lipid. In some examples, the lipid excipients in the gene editing composition may contain about 35 mol% of the cationic lipid. In some examples, the lipid excipients in the gene editing composition may contain about 40 mol% of the cationic lipid. In some examples, the lipid excipients in the gene editing composition may contain about 45 mol% of the cationic lipid. In some examples, the lipid excipients in the gene editing composition may contain about 50 mol% of the cationic lipid. In some examples, the lipid excipients in the gene editing composition may contain about 55 mol% of the cationic lipid. In some examples, the lipid excipients in the gene editing composition may contain about 60 mol% of the cationic lipid. In specific examples, the lipid excipients in the gene editing composition may contain about 47.5 ±20% mol% of the cationic lipid. Lor example, the lipid excipients in the gene editing composition may contain about 47.5 ±15% mol% of the cationic lipid. In yet another specific example, the lipid excipients in the gene editing composition may contain about 47.5 ±10% mol% of the cationic lipid.

[0134] In some examples, the lipid excipients in the HA01 gene editing composition maycomprise about 45-50 mol% of the cationic lipid, for example, about 45-48 mol%, about 46-49 mol%, about 47-48 mol% or about 48-50% of the cationic lipid, based on total mol of the lipid excipients in the LNP. In some examples, the lipid excipients in the gene editing composition may contain about 47.5 ±10% mol% of the cationic lipid. In other examples, the lipid excipients in the gene editing composition may contain about 47.5 ±5% mol% of the cationic lipid. In one specific examples, the lipid excipients in the gene editing composition may contain about 47.5 mol% of the cationic lipid.

[0135] Alternatively, or in addition, the lipid excipients in the HA01 gene editing composition may comprise about 5-15 mol% of the zwitterionic phospholipid, e.g., about 5-15 mol% of the zwitterionic phospholipid, based on total mol of the lipid excipients in the LNP. In some examples, the lipid excipients in the gene editing composition may comprise about 5 mol% of the zwitterionic phospholipid. In some examples, the lipid excipients in the gene editing composition may comprise about 7.5 mol% of the zwitterionic phospholipid. In some examples, the lipid excipients in the gene editing composition may comprise about 10 mol% of the zwitterionic phospholipid. In some examples, the lipid excipients in the gene editing composition may comprise about 12.5 mol% of the zwitterionic phospholipid. In some examples, the lipid excipients in the gene editing composition may comprise about 15 mol% of the zwitterionic phospholipid. In specific examples, the lipid excipients in the gene editing composition may comprise about 10 ±20% mol% of the zwitterionic phospholipid. For example, the lipid excipients in the gene editing composition may comprise about 10 ±15% mol% of the zwitterionic phospholipid. In yet another specific example, the lipid excipients in the gene editing composition may comprise about 10 ±10% mol% of the zwitterionic phospholipid.

[0136] In some examples the lipid excipients in the HA01 gene editing composition may comprise about 9-11 mol% of the zwitterionic phospholipid, for example, about 9-10 mol%, about 9.5-10.5 mol%, or about 10-11 mol% of the zwitterionic phospholipid, based on total mol of the lipid excipients in the LNP. In other examples, the lipid excipients in the gene editing composition may comprise about 10 ±10% mol% of the zwitterionic phospholipid. In yet another specific example, the lipid excipients in the gene editing composition may comprise about 10 ±5% mol% of the zwitterionic phospholipid. In some specific examples, the lipid excipients in the gene editing composition may comprise about 10 mol% of the zwitterionic phospholipid.

[0137] Alternatively, or in addition, the lipid excipients in the HA01 gene editing compositionmay comprise about 25-55 mol% of the cholesterol, for example, about 35-45 mol% of the cholesterol, based on total mol of the lipid excipients in the LNP. In some examples, the lipid excipients in the gene editing composition may comprise about 25 mol% of the cholesterol. In some examples, the lipid excipients in the gene editing composition may comprise about 30 mol% of the cholesterol. In some examples, the lipid excipients in the gene editing composition may comprise about 35 mol% of the cholesterol. In some examples, the lipid excipients in the gene editing composition may comprise about 40 mol% of the cholesterol. In some examples, the lipid excipients in the gene editing composition may comprise about 45 mol% of the cholesterol. In some examples, the lipid excipients in the gene editing composition may comprise about 50 mol% of the cholesterol. In some examples, the lipid excipients in the gene editing composition may comprise about 55 mol% of the cholesterol. In some specific examples, the lipid excipients in the gene editing system may comprise about 40±20% mol% of the cholesterol. For example, the lipid excipients in the gene editing system may comprise about 40±15% mol% of the cholesterol. In another specific example, the lipid excipients in the gene editing system may comprise about 40±10% mol% of the cholesterol.

[0138] In some examples, the lipid excipients in the HA01 gene editing composition may comprise about 38-42 mol% of the cholesterol, for example, about 38-40 mol%, about 39-41 mol%, about 39.5-41.5 mol%, or about 40-42 mol% of the cholesterol, based on total mol of the lipid excipients in the LNP. In some examples, the lipid excipients in the gene editing system may comprise about 40±10% mol% of the cholesterol. In other examples, the lipid excipients in the gene editing system may comprise about 40±5% mol% of the cholesterol. In one specific example, the lipid excipients in the gene editing system may comprise about 40 mol% of the cholesterol.

[0139] Alternatively, or in addition, the lipid excipients in the HA01 gene editing composition may comprise about 0.5-5 mol% of the pegylated lipid, for example, about 1-5 mol% of the pegylated lipid, based on total mol of the lipid excipients in the LNP. In some examples, the lipid excipients in the gene editing composition may comprise about 0.5 mol% of the pegylated lipid. In some examples, the lipid excipients in the gene editing composition may comprise about 1.0 mol% of the pegylated lipid. In some examples, the lipid excipients in the gene editing composition may comprise about 1.5 mol% of the pegylated lipid. In some examples, the lipid excipients in the gene editing composition may comprise about 2.0 mol% of the pegylated lipid. In some examples, the lipid excipients in the gene editing composition may comprise about 2.5 mol% of the pegylatedlipid. In some examples, the lipid excipients in the gene editing composition may comprise about 3.0 mol% of the pegylated lipid. In some examples, the lipid excipients in the gene editing composition may comprise about 3.5 mol% of the pegylated lipid. In some examples, the lipid excipients in the gene editing composition may comprise about 4.0 mol% of the pegylated lipid. In some examples, the lipid excipients in the gene editing composition may comprise about 4.5 mol% of the pegylated lipid. In some examples, the lipid excipients in the gene editing composition may comprise about 5.0 mol% of the pegylated lipid. In some specific examples, the lipid excipients in the gene editing composition may comprise 2.5±20% mol% of the pegylated lipid. For example, the lipid excipients in the gene editing compositions may comprise about 2.5±15% mol% of the pegylated lipid. In another specific example, the lipid excipients in the gene editing compositions may comprise about 2.5±10% mol% of the pegylated lipid.

[0140] In some examples, the lipid excipients in the HAO1 gene editing composition may comprise about 2.2-2.8 mol% of the pegylated lipid, for example, about 2.2-2.5 mol%, about 2.4-2.6 mol%, or about 2.6-2.8 mol% of the pegylated lipid, based on total mol of the lipid excipients in the LNP. In some examples, the lipid excipients in the gene editing composition may comprise 2.5±10% mol% of the pegylated lipid. For example, the lipid excipients in the gene editing compositions may comprise about 2.5±5% mol% of the pegylated lipid. In some specific examples, the lipid excipients in the gene editing compositions may comprise about 2.5 mol% of the pegylated lipid.

[0141] In one specific example, the lipid excipients contain about 47.5 mol% of the cationic lipid (e.g., CL- A), about 10 mol% of the zwitterionic phospholipid (e.g., DSPC), about 40 mol% of cholesterol, and about 2.5 mol% of the PEG lipid (e.g., PL- A).

[0142] The N / P ratio of the LNPs, where N represents the moles of cationic lipid and P represents the moles of phosphate present as part of the nucleic acid backbone, ranges from 2:1 to 30:1, for example 3:1 to 22:1. In other embodiments, N / P ranges from 6: 1 to 20:1, 3:1 to 9:1 or 2:1 to 12:1. Exemplary N / P ranges include about 3:1, about 6:1, about 9:1, about 12:1 and about 22:1.

[0143] I). Exemplary HAO1 Gene Editing Compositions

[0144] In some embodiments, the HA01 gene editing composition provided herein comprises a mRNA molecule encoding a Casl2i2 polypeptide as disclosed herein (e.g., SEQ ID NO: 7 or SEQ ID NO: 17), a RNA guide comprising a spacer sequence specific to SEQ ID NO: 10 in the exon 1of human HA01 gene and a direct repeat sequence of SEQ ID NO: 12, and lipid excipients, which may form LNPs associated with the mRNA and / or gRNA components in the gene editing composition. In some examples, the mRNA molecule may comprise a nucleotide sequence at least 70% (e.g., at least 80%, at least 85%, at least 90%, at least 95% or above) identical to SEQ ID NO: 8 and encode the amino acid sequence of SEQ ID NO: 7. In one specific example, the mRNA may comprise the nucleotide sequence of SEQ ID NO: 8.

[0145] Any of the specific Casl2i2 polypeptides (e.g., SEQ ID NO: 7 or SEQ ID NO: 17), as well as their encoding nucleic acids (e.g., those provided in the Sequence Table below) are within the scope of the present disclosure.

[0146] In the lipid excipients, the cationic lipid can have the structure of

[0147]

[0148] or is a pharmaceutically acceptable salt or stereoisomer thereof. The pegylated lipid can have the

[0149]

[0150] structure of , in which the average n is between 45-49, inclusive (e.g., 45, 46, 47, 48, or 49). The zwitterionic phospholipid is l,2-Distearoyl-sn-glycero-3 -phosphocholine (DSPC).

[0151] In the exemplary HAO1 gene editing composition, the LNPs may comprise 47.5 ±20% mol% of the cationic lipid, 10 ±20% mol% of the zwitterionic phospholipid, 40±20% mol% of the cholesterol, and 2.5±20% mol% of the pegylated lipid, based on total mol of the lipid excipients in the LNP. In some examples, the LNPs may comprise 47.5 ±15% mol% of the cationic lipid, 10 ±15% mol% of the zwitterionic phospholipid, 40±15% mol% of the cholesterol, and 2.5±15%mol% of the pegylated lipid, based on total mol of the lipid excipients in the LNP. In other examples, the LNPs may comprise 47.5 ±10% mol% of the cationic lipid, 10 ±10% mol% of the zwitterionic phospholipid, 40±10% mol% of the cholesterol, and 2.5±10% mol% of the pegylated lipid, based on total mol of the lipid excipients in the LNP. In one specific example, the LNPs may comprise about 47.5 mol% of the cationic lipid, about 10 mol% of the zwitterionic phospholipid, about 40 mol% of the cholesterol, and about 2.5 mol% of the pegylated lipid, based on total mol of the lipid excipients in the LNP.

[0152] In specific examples, the exemplary HA01 gene editing composition may comprise the LNPs, which may comprise 45-50 mol% (e.g., about 47.5 mol%) of the cationic lipid, 9-11 mol% (e.g., about 10) mol% of the zwitterionic phospholipid, 38-42 mol% (e.g., about 40 mol%) of the cholesterol, and 2.2-2.8 (e.g., about 2.5) mol% of the pegylated lipid, based on total mol of the lipid excipients in the LNP. In one specific example, the LNPs may comprise about 47.5 mol% of the cationic lipid, about 10 mol% of the zwitterionic phospholipid, about 40 mol% of the cholesterol, and about 2.5 mol% of the pegylated lipid, based on total mol of the lipid excipients in the LNP.

[0153] In some examples, the mRNA comprises UTRs flanking the coding sequence for the Casl2i2 polypeptide, for example, a 5’ UTR and a 3’ UTR and a poly A tail. In one specific example, the mRNA may comprise (e.g., consists of) the nucleotide sequence of SEQ ID NO: 23. Further, the mRNA may carry a 5’ cap, e.g, m7G(5')ppp(5')(2'OMeA)pG, and may contain pseudouridine residues in replacement of uridine residues (e.g, 100%).

[0154] In some examples, the RNA guide may comprise a spacer sequence of SEQ ID NO: 11. Such an RNA guide may comprise (e.g., consists of) the nucleotide sequence of SEQ ID NO: 13. The RNA guide may comprise one or more modifications, e.g., phosphothioate linkage and 2’-O-methylation, which may be located at the 5’ and / or 3’ nucleotides. In one specific example, the RNA guide is set forth in SEQ ID NO: 14.

[0155] In some instances, the HA01 gene editing composition provided herein comprises (a) a mRNA molecule encoding a Casl2i2 polypeptide comprising the amino acid sequence of SEQ ID NO: 7, (b) a RNA guide comprising the spacer of SEQ ID NO: 11 and a direct repeat sequence of SEQ ID NO: 12, and (c) lipid excipients, which may form LNPs associated with the mRNA and / or gRNA components in the gene editing composition. In some examples, the mRNA may comprise a nucleotide sequence at least 70% (e.g, at least 80%, at least 85%, at least 90%, at least 95% orabove) identical to SEQ ID NO: 8 and encodes the Casl2i2 polypeptide of SEQ ID NO: 7. In specific examples, the mRNA may comprise the nucleotide sequence of SEQ ID NO: 23. In some examples, the gRNA may comprise the nucleotide sequence of SEQ ID NO: 13, for example, a modified gRNA set forth in SEQ ID NO: 14.

[0156] In some examples, the mRNA and RNA guide have a ratio of l±20% (e.g., 0.8 to 1.2) in theHAOl gene editing composition. In some specific examples, the ratio is 1:1 (1.0).

[0157] In some examples, the HAO1 gene editing composition has a nitrogemphosphate (N / P) ratio of 6.0 ± 20% (e.g., 6.0±15%, 6.0±10%, 6.0±5%, 6.0±2%, or 6.0±l%), which represents the relative amount LNPs and nucleic acids in the composition. In other examples, the HAO1 gene editing composition has a nitrogen: phosphate (N / P) ratio of 6.0 ± 1 (e.g., 6.0±0.5). In one example, the N / P ratio in the HAO1 gene editing composition provided herein ranges from 5.18 to 7 (e.g., 5.6 or 6.0).

[0158] In general, the N / P ratio may be calculated from the results of two assays, HPLC (for determination of aminolipid mol) and ribogreen (for determination of mol of total RNA). See, e.g., PCT / US2025 / 010228, filed on January 3rd, 2025, the relevant disclosures of which are incorporated by reference for the subject matter and purposes referenced herein.

[0159] IL Preparation of Gene Editing Components

[0160] The present disclosure provides methods for production of components of the gene editing systems disclosed herein, e.g., the RNA guide, methods for production of the Casl2i polypeptide-coding mRNA, and methods for complexing the RNA guide and Casl2i mRNA with the lipid excipients.

[0161] A. RNA Guides and C as 12i2 -Encoding mRNAs

[0162] In some embodiments, the RNA guide is made by in vitro transcription of a DNA template. Thus, for example, in some embodiments, the RNA guide is generated by in vitro transcription of a DNA template encoding the RNA guide and / or the mRNA using an upstream promoter sequence (e.g., a T7 polymerase promoter sequence). In some embodiments, the DNA template encodes multiple RNA guides or the in vitro transcription reaction includes multiple different DNA templates, each encoding a different RNA guide. In some embodiments, the DNA template encodes both the RNA guide and the mRNA molecule and can produce both in vitro as separateRNA molecules.

[0163] In some embodiments, the mRNA is made by in vitro transcription of a DNA template. In some examples, the mRNA made by in vitro transcription may be subject to modifications such as addition of a 5 ’-cap (e.g., those disclosed herein), following methods known in the art. In some embodiments synthetic caping residues, synthetic 5 ’-caps (e.g., those disclosed herein) may be used in the in vitro transcription so that the mRNA products comprise a synthetic 5 ’-cap. Alternatively, or in addition, modified nucleotide residues (e.g., pseudouridine residues) may be used in the in vitro transcription so that the modified nucleotide residues are incorporated into the mRNA products thus produced.

[0164] In some embodiments, the RNA guide is made using chemical synthetic methods. In some examples, the RNA guide can be synthesized using one or more modified nucleotide, e.g, as described above.

[0165] B. Nucleic Acid-Loaded LNPs

[0166] In some embodiments, the HA01 gene editing compositions disclosed herein comprise lipid excipients (e.g., those disclosed herein), which form LNPs, and the nucleic acid components in the composition (mRNA and gRNA) are attached to or encapsulated by the LNPs, resulting in nucleic acid-loaded LNPs.

[0167] A variety of methods are available for preparing LNPs. See, e.g, Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,946,787, PCT Publication No. WO 91 / 17424, Deamer & Bangham, Biochim. Biophys. Acta 443:629-634 (1976); Fraley, et al., PNAS 76:3348-3352 (1979); Hope et al., Biochim. Biophys. Acta 812:55-65 (1985); Mayer et al., Biochim. Biophys. Acta 858:161-168 (1986); Williams et al., PNAS 85:242-246 (1988); Hope et al., Chem. Phys. Lip. 40:89 (1986); Gregoriadis, Liposome Technology (1984). Such conventional methods can be used to prepare the LNPs with the lipid excipients disclosed herein. Any of such methods may be performed in the presence of the nucleic acid components of the gene editing composition (mRNA and gRNA) such that the resultant lipid nanoparticles would carry the nucleic acids to produce nucleic acid-loaded LNPs.

[0168] The lipid nanoparticles prepared following any of the methods known in the art or disclosedherein can be analyzed to determine concentration and / or particle size distribution (e.g., by NTA). Alternatively, or in addition, the lipid nanoparticles can be fractionated and particles having suitable sizes may be collected for use in the fusion method disclosed herein.

[0169] In addition, the nucleic acid-loaded LNPs may be analyzed to determine the N / P ratio following methods known in the art and / or disclosures provided herein. The N / P ratio is the ratio of moles of cationic lipid (N) to moles of phosphate present as part of the nucleic acid backbone (P).

[0170] LNPs can be prepared according to methods known in the art, for example as disclosed in W02020 / 061426 and W02021 / 030701, the relevant disclosures of which are incorporated herein by reference in its entirety. Briefly, cationic lipid, zwitterionic phospholipid, cholesterol and pegylated are solubilized in ethanol at a molar ratio of 40-50:5-15:35-45: 1-5, respectively. LNPs are generally prepared at a total lipid to mRNA weight ratio of approximately 10: 1 to 30: 1. mRNA is diluted to 0.2 mg / mL in 10 to 50 mM citrate buffer, pH 4. Syringe pumps are used to mix the ethanolic lipid solution with the mRNA aqueous solution at a ratio of about 1:5 to 1:3 (vol / vol) with total flow rates above 15 ml / min. The ethanol is then removed and the external buffer replaced with PBS by dialysis. Finally, the lipid nanoparticles ae filtered through a 0.2 pm pore sterile filter.

[0171] III. Methods for Genetic Editing of HAO1 Gene and Treatment of Primary Hyperoxaluria 1

[0172] Any of the HA01 gene editing compositions disclosed herein can be used to genetically edit the HA01 gene in host cells. In some embodiments, the methods comprise introducing the HA01 gene editing composition into host cells. Such an HA01 gene editing composition may comprise nucleic acid (mRNAZgRNA)-loaded LNPs as disclosed herein. In some instances, the HA01 gene editing composition can be introduced into in vv / r -cultured host cells. In other instances, the HA01 gene editing composition described herein is delivered to a subject to genetically modify the HA01 gene in the subject.

[0173] As used herein, the term “edit” refers to one or more modifications introduced into a target nucleic acid, e.g., within the HA01 gene. The edit can be one or more substitutions, one or more insertions, one or more deletions, or a combination thereof. As used herein, the term “substitution” refers to a replacement of a nucleotide or nucleotides with a different nucleotide or nucleotides, relative to a reference sequence. As used herein, the term “insertion” refers to a gain of a nucleotideor nucleotides in a nucleic acid sequence, relative to a reference sequence. As used herein, the term “deletion” refers to a loss of a nucleotide or nucleotides in a nucleic acid sequence, relative to a reference sequence.

[0174] Any of the HA01 gene editing compositions as disclosed herein (e.g., comprising nucleic acid-loaded LNPs) or modified cells generated using such a gene editing system as disclosed herein may be used for treating a disease that would benefit from genetic editing of the HA01 gene, for example, primary hyperoxaluria 1 (PHI).

[0175] PH is a rare genetic disorder effecting subjects of all ages from infants to elderly. PH includes three subtypes involving genetic defects that alter the expression of three distinct proteins. PHI involves alanine-glyoxylate aminotransferase, or AGT / AGT1.

[0176] In PHI, excess oxalate can also combine with calcium to form calcium oxalate in the kidney and other organs. Deposits of calcium oxalate can produce widespread deposition of calcium oxalate (nephrocalcinosis) or formation of kidney and bladder stones (urolithiasis) and lead to kidney damage. Common kidney complications in PHI include blood in the urine (hematuria), urinary tract infections, kidney damage, and end-stage renal disease (ESRD). Over time, kidneys in patients with PHI may begin to fail, and levels of oxalate may rise in the blood. Deposition of oxalate in tissues throughout the body, e.g., systemic oxalosis, may occur due to high blood levels of oxalate and can lead to complications in bone, skin, and eye. Patients with PHI normally have kidney failure at an early age, with renal dialysis or dual kidney / liver organ transplant as the only treatment options.

[0177] In some embodiments, provided herein is a method for treating PH such as PHI in a subject (e.g., a human patient) in need of the treatment. The method may comprise administering to the subject (e.g., the human patient) in need of the treatment any of the HA01 gene editing composition disclosed herein. The HA01 gene editing composition may be delivered to a specific tissue or specific type of cells where the gene edit is needed. In some examples, the HA01 gene editing composition comprises LNPs loaded with the Casl2i2-encoding mRNA and the HA01-targeting gRNA as also disclosed herein. In specific examples, the exemplary HA01 gene editing composition disclosed above can be used in the treatment method disclosed herein.

[0178] The HA01 gene editing composition disclosed herein may be prepared, packaged, or sold in a formulation for a suitable administration route, for example, intravenous or another route of administration. The composition of the disclosure may be prepared, packaged, or sold in bulk, asa single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is a discrete amount of the pharmaceutical composition (e.g., the gene editing system or components thereof), which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. Unless explicitly stated, the doses of the HA01 gene editing composition disclosed herein refer to the amount of total RNA (i.e.: mRNA+gRNA) in the gene editing composition.

[0179] In some embodiments, the HA01 gene editing composition as disclosed herein may be administered to a subject in need thereof, e.g., one who suffers from a liver disease associated with the HA01 gene. In some instances, the gene editing composition may be delivered to specific cells or tissue (e.g. , to liver cells), where the gene editing components could function to genetically modify the HA01 gene in such cells.

[0180] A formulation of the HA01 gene editing composition suitable for parenteral administration may comprise the active agent (e.g., the mRNA encoding the nuclease and gRNA) combined with a pharmaceutically acceptable excipients (e.g., the lipid excipients such as the LNPs formed thereby) and carrier, such as sterile water or sterile isotonic saline. Such a formulation may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Some injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Some formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Some formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.

[0181] The HA01 gene editing composition may be in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the cells, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulation may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or saline. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which that are useful include those which may comprise the cells in a packaged form, in a liposomal preparation, or as a component of a biodegradable polymer system.Some compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0182] In some embodiments, an effective amount of the HA01 gene editing composition disclosed herein (e.g., the exemplary HA01 gene editing composition disclosed herein) can be administered to a human patient having PH (e.g., PHI) via a suitable route, for example, intravenous infusion, to treating the disease.

[0183] As used herein, the term “treating” refers to the application or administration of a composition including one or more active agents (here the nuclease-encoding mRNA and the HAO 1 -targeting gRNA) to a subject, who has a target disease or disorder (PH such as PHI in the instant disclosure), a symptom of the disease / disorder, or a predisposition toward the disease / disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward the disease or disorder.

[0184] Alleviating a target disease / disorder includes delaying the development or progression of the disease, or reducing disease severity or prolonging survival. Alleviating the disease or prolonging survival does not necessarily require curative results. As used therein, "delaying" the development of a target disease or disorder means to defer, hinder, slow, retard, stabilize, and / or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or individuals being treated. A method that “delays” or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and / or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

[0185] “Development” or “progression” of a disease means initial manifestations and / or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of a target disease or disorder includes initialonset and / or recurrence.

[0186] As used herein, “an effective amount” refers to the amount of each active agent (e.g., the nuclease-encoding mRNA and the HO Al -targeting gRNA) required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents. Determination of whether an amount of the HA01 gene editing composition the therapeutic effect would be evident to one of skill in the art. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.

[0187] Empirical considerations, such as the half-life, generally will contribute to the determination of the dosage. Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and / or suppression and / or amelioration and / or delay of a target disease / disorder.

[0188] In one example, dosages for the HA01 gene editing composition, specifically the mRNA and gRNA components therein, as described herein may be determined empirically in individuals who have been given one or more administration(s) of the composition. Individuals are given incremental dosages of the agonist. To assess efficacy of the agonist, an indicator of the disease / disorder can be followed.

[0189] In some embodiments, a human PH patient can be administered the HA01 gene editing composition disclosed herein at a dose of about 2.0-5.0 mg of the nucleic acids in the composition (mRNA and gRNA in total) per kg of the patient’s body weight.

[0190] In some embodiments, the subject to be treated by any of the HA01 gene editing composition as disclosed herein may be a human patient diagnosed with PHI, e.g., via routine medical practice. In some example, PHI can be determined by genetic analysis confirming pathogenic mutations in the alanine-glyoxylate aminotransferase (AGXT) gene. The human patients for treatment may meet one or more (e.g., all) inclusion and exclusion criteria listed in Example 2 below.In some instances, the human patient may be an adult patient (e.g., 18-64 years, inclusive). In other instances, the human patient may be a pediatric patient (e.g., 6 to 17 years, inclusive). The human patient may have a mean of 2 valid 24-hour urinary oxalate excretion (UOx) >0.7 mmol / 24 hours / 1.73m2, a preserved kidney function at estimated glomerular filtration rate (eGFR) > 30 mL / mim / 1.73m2, or a combination thereof. In some instances, the human patient may have a body weight < 90 kg. In some instances, the human patient does not exhibit evidence of systemic oxalosis.

[0191] Alternatively, or in addition, the subject to be treated by any of the HA01 gene editing composition as disclosed herein may be a human patient who has no confirmed diagnosis of primary hyperoxaluria type 2 or type 3. In some instances, the human patient has no history of a liver, kidney, or combined liver / kidney transplant. Alternatively, or in addition, the human patient is not currently on dialysis, or anticipated requirement for dialysis within the initial 24 months of the treatment. In some instances, the human patient may not have participated in previous use (e.g., within 24 months prior to the treatment), or is not currently receiving urinary oxalate lowering RNA interference (RNAi) or siRNA therapy. Alternatively, the human patient has received RNAi or siRNA therapy prior to the treatment by the gene editing system disclosed herein. In some examples, the human patient may receive RNAi or siRNA therapy after the treatment by the gene editing system disclosed herein. In some instances, the human patient is free of liver cirrhosis history.

[0192] In some embodiments, a human patient subject to the treatment may receive pre-treatment involving steroid and / or antihistamine prior to administration of the HA01 gene editing composition. Such pre-treatment may comprise oral dexamethasone (adult dose 8 mg; pediatric dose 0.15 mg / kg up to maximum 8 mg) 16-24 hours prior to the start of the administration of the gene editing composition. Further, the human patient may receive one or more of the following pre-medi cations about 1-2 hours prior to the HA01 gene editing composition:

[0193] • Intravenous dexamethasone (adult dose 8 or 10 mg; pediatric dose 0.18 mg / kg up to maximum dose 8 or 10 mg).

[0194] • Intravenous Hi blocker (adult dose diphenhydramine 50 mg or equivalent; pediatric dose 2 mg / kg up to maximum 50 mg) or oral Hi blocker (adult and pediatric dose cetirizine 10 mg or equivalent).

[0195] • Intravenous or oral H2 blocker (adult dose famotidine 20 mg or equivalent; pediatricdose 0.25 mg / kg or equivalent).

[0196] • IV acetaminophen / paracetamol 15 mg / kg (<50 kg) or 750 mg (>50 kg).

[0197] In some embodiments, the human patient, optionally after the pre-treatment and pre-medications, may be given an effective amount of the gene editing composition (represented by the amount of total RNA) by a suitable administration route, for example, intravenous infusion. The human patient may receive a single dose of the gene editing composition. In some instances, the human patient may receive more than one dose. In some examples, the human patient may receive 2 doses of the gene editing composition. In other examples, the human patient may receive 3 doses of the gene editing composition. In some instances, the gene editing composition is administered to the human patient via intravenous infusion, for example, over 2 hours. In some instances, the gene editing composition is administered to the human patient via intravenous infusion, for example, for 3, 4, 5, 6, 7, or 8 hours. In some instances, the gene editing composition is administered to the human patient via intravenous infusion, for example, for 2-3, 2-4, 2-5, 2-6, 2-7 or 2-8 hours. In some instances, the gene editing composition is administered to the human patient via intravenous infusion, for example, for 3-4, 3-5, 4-5, 4-6, 5-6, 5-7, 6-7 or 6-8 hours. In some instances, the gene editing composition is administered to the human patient via intravenous infusion for about 2 hours. In some instances, the dose of the gene editing composition may range from about 0.4 mg / kg to about 1 mg / kg. In some examples, the dose of the gene editing composition may range from about 0.4 mg / kg to about 0.5 mg / kg, about 0.5 mg / kg to about 0.6 mg / kg, about 0.6 mg / kg to about 0.7 mg / kg, about 0.7 mg / kg to about 0.8 mg / kg, about 0.8 mg / kg to about 0.9 mg / kg, or about 0.9 mg / kg to about 1.0 mg / kg. In one example, the dose of the gene editing composition can be 0.4 mg / kg ±10% (e.g., 0.4 mg / kg). In another example, the dose of the gene editing composition can be 0.7 mg / kg ±10% (e.g., 0.7 mg / kg). In yet another example, the dose of the gene editing composition can be 1.0 mg / kg ±10% (e.g., 1.0 mg / kg). The dose of the gene editing composition may not exceed 75 mg, regardless of the patient’s body weight.

[0198] In some embodiments, the human patient may receive post-treatment after receiving the gene editing composition, for example, intravenous dexamethasone (adult dose of 4 mg; pediatric dose of 0.075 mg / kg up to a dose of 4 mg / kg) at 6 hours and 24 hours after completion of the gene editing composition treatment.

[0199] In some embodiments the human patient is monitored for occurrence of adverse events, as well as other physical features such as physical examination, height, weight, vital signs, 12-leadelectrocardiograms, clinical laboratory tests (including hematology, serum chemistry, coagulation parameters, inflammatory markers, urinalysis, urine and serum pregnancy tests, eGFR) before and / or after the gene editing treatment as safety measures. Exemplary adverse events include allergic bronchospasm, blood dyscrasias or convulsions, infusion-related reactions (e.g., moderate to severe), acute liver injury (e.g., evidenced by CTCAE > Grade 2 elevation in AST, ALT, total bilirubin, or clinically relevant symptoms and / or signs of liver injury), abnormal coagulation, muscle injury, events related to spleen, event related to adrenal gland issues, hyperglycemia, or a combination thereof. See Example 2 below for details. Once an adverse event is observed, management of such shall be performed to the human patient following routine medical practice.

[0200] Treatment efficacy can be evaluated via routine medical practice. In some embodiments, the host cells and / or the subject treated by the HA01 gene editing composition may be examined for genetic edits in the HA01 gene, for example, percentage of Indels in the HA01 gene (e.g., at the target sequence site). Alternatively, or in addition, HA01 gene product (the GO enzyme) and molecules involved in the pathway catalyzed by the GO enzyme, e.g., glycolate, glyoxylate, and / or oxalate can be measured in the subject before and after treatment for assessing HA01 gene editing outcome.

[0201] V. Kits and Uses Thereof

[0202] The present disclosure also provides kits that can be used, for example, to carry out a method described herein for genetical modification of the HA01 gene. In some embodiments, the kits include the components of the HA01 gene editing composition disclosed herein, e.g., the mRNA encoding a Casl2i2 polypeptide, the gRNA targeting the HA01 gene, and the lipid excipients. In some embodiments, the kit comprises LNPs loaded with the mRNA and gRNA. The kits can additionally include, optionally, a buffer and / or instructions for use of the HA01 gene editing composition.

[0203] In some embodiments, the kit can comprise instructions for use in accordance with any of the methods described herein. The included instructions can comprise a description of administration of the HA01 gene editing composition disclosed herein to treat, delay the onset, or alleviate the target disease as also described herein (PH such as PHI). The kit may further comprise a description of selecting an individual suitable for treatment based on identifying whether that individual has the target disease.The instructions relating to the use of the HA01 gene editing composition generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or subunit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0204] The label or package insert indicates that the composition is used for treating, delaying the onset and / or alleviating the target disease. Instructions may be provided for practicing any of the methods described herein.

[0205] The kits of this invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. A kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).

[0206] Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the present disclosure provides articles of manufacture comprising contents of the kits described above.

[0207] SEQUENCE TABLE

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219] • 2'-0-methyl phosphorothioate modifications indicated: *, phosphorothioate; m, 2’ O-methyl

[0220] • Underlined and italicized nucleotide sequences refer to 5’ and 3’ untranslated regions (UTRs)

[0221] • “n” refers to adenosine (a) residues or is absent.

[0222] General techniques

[0223] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of theart. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan etal., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.(1985»; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984»; Animal Cell Culture (R.I. Freshney, ed. (1986»; Immobilized Cells and Enzymes (IRL Press, (1986»; and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.).

[0224] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.EXAMPLES

[0225] The following examples are provided to further illustrate some embodiments of the present invention but are not intended to limit the scope of the invention; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

[0226] Example 1: Efficacy and Tolerability of HAO1 Gene Editing System in Non-Human Primates

[0227] This example evaluates the pharmacologic activity and efficacy of the HAO 1 -targeting gene editing system disclosed herein, comprising a Casl2i2 mRNA (SEQ ID NO: 23) and a HAO 1 -targeting gRNA (SEQ ID NO: 14) loaded into lipid nanoparticles in a non-human primate (NHP) cynomolgus macaque model. A single dose 8-week study was performed, in which the gene editing system was administered to cynomolgus monkeys via intravenous infusion.

[0228] Study Design

[0229] Cynomolgus monkeys (2-3 / sex / group) were intravenously (IV) administered a single dose of control article (Dulbecco’s phosphate-buffered saline [DPBS], 300 mM sucrose, pH

[0230] 7.4, in 0.9% sodium chloride injection, United States Pharmacopeia (USP) [saline]) or 2.25, 2.5, or 3.0 mg / kg of the HAO 1 -targeting gene editing system disclosed herein. Serum and liver samples were collected from the animals for glycolate and glycolate oxidase (GO) enzyme activity, respectively, to assess pharmacologic activity. Serum samples collected from the pretest period (Day -11 or -12) and after dosing on Days 8, 15, 22, 29, 43, and 56 were analyzed for glycolate using gas chromatography-mass spectrometry (GC-MS). Samples from the

[0231] caudal and left liver lobes of each animal were collected at necropsy on Day 56 and analyzed for GO enzyme activity using a high-pressure liquid chromatography (HPLC)-based approach.

[0232] Table 1 summarizes the experimental design.

[0233] Table 1. Experimental Design

[0234]

[0235] Results

[0236] Determination of Serum Glycolate Levels

[0237] Serum glycolate was measured by Metabolic Solutions using a qualified method (Metabolic Solution Study No. 1894, 2023). Briefly, a known amount of13C2-glycolate (10 pM) was added to each 50 pL serum sample. Serum proteins were precipitated by acetonitrile, removed by centrifugation at room temperature, and the deproteinized serum was evaporated to dryness by nitrogen gas. Methoxylamine hydrochloride in pyridine was reacted first with glycolate for 20 minutes at 100°C. Next, N-methyltrimethylsilyltrifluoroacetamide (MSTFA) was added and heated for 20 minutes at 100°C to form the trimethylsilyl (TMS) derivative of glycolate. The TMS-glycolate derivative was determined by GC-MS using positive chemical ionization detection using an Agilent 5977 EI / CI MSD with an Agilent 7890 GC. A Phenomenex ZB-5HT Plus capillary column was used to separate the TMS glycolate derivative. Selected ion chromatograms were obtained by monitoring ions m / z 221 and 223 for glycolate and13C2-glycolate, respectively. Concentration of glycolate in micromoles per liter (pM) was calculated from peak area ratios and a concentration calibration curve.

[0238] For each animal and at each time point, the concentration of serum glycolate (pM) and fold change (FC) in glycolate from pretest (also referred to as baseline) was calculated. Statistically significant differences in glycolate levels and FC glycolate between each gene editing composition-treated group and control article group were determined using 1-way analysis of variance (ANOVA) test with Dunnett’s multiple comparisons test.

[0239] Sex-combined group mean serum glycolate levels were significantly higher on Day 8 through Day 56 after a single dose of the gene editing composition at the indicated dose to cynomolgus monkeys compared with control-dosed animals (FIGs. 1A). When separated by sex, the same general trend in significance across evaluated study days was observed for both sexes at 3 mg / kg. At 2.5 mg / kg, group mean serum glycolate levels were not significantly higher, but were elevated compared with control for males and were similar to control for females. Levels of serum glycolate were not significantly higher compared with control for 2.25 mg / kg of the gene editing system both with sexes combined or separated. In general serum glycolate levels reached a plateau by Day 43, with similar levels between Day 43 and Day 56. See FIGs. 1B-1C.The fold change (FC) in serum glycolate for each group compared with pretest (baseline) values was also evaluated. After a single dose of 2.5 and 3 mg / kg of the gene editing system, there was a distinct increase in serum glycolate beginning on Day 8 or 15 which persisted out to Day 56. The average FC ± standard deviation (SD) in serum glycolate at 2.5 and 3 mg / kg of the gene editing composition was as high as 2.1 ± 0.9 and 2.4 ± 0.9, respectively, across the study. These FC increases reached statistical significance for several timepoints, including end of study (Day 56). When separated by sex, the FC in serum glycolate was higher for males compared with females at 2.5 (as high as 2.9±0.4 and 1.6±0.8, respectively) and 3 mg / kg gene editing composition (as high as 2.6±0.2 and 2.2±1.44, respectively). FC increases were statistically significant for males at doses > 2.5 mg / kg for some timepoints, but not for females. For both sexes, there were minimal changes in FC serum glycolate at 2.25 mg / kg gene editing composition (group mean <1.2-fold and <1.4-fold for males and females, respectively). In general serum glycolate levels reached a plateau by Day 43, with similar levels between Day 43 and Day 56. See FIGs. 1D-1F.

[0240] Determination of GO Enzyme Activity

[0241] GO enzyme activity was measured using an HPLC-based approach which measures glyoxylate levels after the addition of sodium glycolate substrate to liver tissue homogenates. In this assay, 25-50 mg of tissue was sonicated in a lysis buffer containing HEPES and 10% Triton-X. Liver lysate solution was incubated with sodium glycolate master mix (100 mM Tris HC1], 5 mMNa glycolate) for 15 minutes at 37°C. After incubation, 5M perchloric acid (PC A) was added, samples were centrifuged for 5 minutes, and the supernatants were run on HPLC to detect glyoxylate levels. For the readout of this assay, HAO 1 -edited samples were expected to have lower glyoxylate levels as GO (the enzyme encoded by the HA01 gene) is responsible for the conversion of glycolate to glyoxylate. Before running any assay samples, 0.5M PCA blank (derivatized) and 2x 1 pM of glyoxylate / pyruvate standard (derivatized) were injected into the HPLC to ensure performance as expected.

[0242] GO enzyme activity (nmol / minute / mg of liver tissue) was measured from each animal’s liver samples (left liver lobe and caudal liver lobe) at Day 56. GO enzyme activity is reported for both sections of liver per animal as well as the average of these samples. Statistically significant differences in liver GO enzyme activity between each gene editing composition-treated group and control article group were determined using a 1-way ANOVA with Dunnett’s multiplecomparisons test. Percent GO enzyme activity for each individual animal was calculated by dividing each individual animal value by the control group average.

[0243] GO enzyme activity levels were also correlated with liver tissue editing of HA01 (measured by indel analysis reported in Study 2093.05) by simple linear regression using 95% confidence intervals.

[0244] There was a dose-dependent, significant reduction in group mean liver GO enzyme activity at all doses of the gene editing composition compared to control dosed animals at Day 56 (Figure 2). The percent GO enzyme activity in liver compared to control (±SD) was 49.5 ± 9.8, 36.2 ± 14.1, and 19.0 ± 15.1 at 2.25, 2.5, and 3.0 mg / kg of the gene editing composition, respectively. This same dose-dependent reduction in group mean GO enzyme activity was also seen when separated by sex, although for females the level of GO enzyme activity was similar at 2.25 and 2.5 mg / kg of the gene editing system. The mean percent GO enzyme activity in liver compared to control was 42.7 ± 17.9, 23.1 ± 13.6, and 15.1± 8.7 for males and 56.4 ± 15.7, 49.3 ± 11.7, and 22.9± 22.8 for females at 2.25, 2.5, and 3.0 mg / kg of the gene editing composition, respectively.

[0245] Determination of Serum Glycolate, GO Enzyme Activity, and HAO1 Editing in Liver Both reductions in GO enzyme activity and resultant increases in serum glycolate levels demonstrate pharmacologic activity of the gene editing composition via loss of function of HAO 1 gene. The levels of liver editing of HAO 1 as measured by percent indels was compared to each animal’s GO enzyme activity at Day 56 (FIG. 3). A strong relationship between dose of the gene editing system, liver HA01 editing, and GO enzyme activity is shown, with higher levels of editing generally corresponding to higher doses of the gene editing system and lower GO enzyme activity (R2= 0.8592). A tabulated comparison of Day 56 serum glycolate, Day 56 liver GO enzyme activity, and percent liver indels at HA01 is presented in Table 2.

[0246] Table 2. Summary of Pharmacologic Activity of Gene Editing Composition in Cynomolgus Monkeys

[0247]

[0248]

[0249] Conclusion

[0250] Pharmacologic activity of the HAO 1 -targeting gene editing system provided herein was demonstrated in cynomolgus monkeys after a single IV dose. Increases in serum glycolate were apparent at doses > 2.5 mg / kg of the gene editing system, while a dose-dependent reduction in GO enzyme activity was seen at 2.25, 2.5, and 3.0 mg / kg of the gene editing system. In general, pharmacologic activity between sexes were similar at 2.25 and 3.0 mg / kg of the gene editing system, while at 2.5 mg / kg, increases in serum glycolate and reductions in GO enzyme activity were higher for males compared with females. A strong relationship between dose of the gene editing system, HA01 editing, and GO enzyme activity was demonstrated 8 weeks after administration.

[0251] Example 2: A Phase 1 / 2 Dose Escalation Study to Evaluate the Safety, Tolerability,

[0252] Pharmacokinetics, Pharmacodynamics and Efficacy of a Gene Editing System Targeting HAO1 in Participants with Primary Hyperoxaluria Type 1 (PHI) This example illustrates a human Phase 1 / 2 clinical trial to investigate safety, tolerability, pharmacokinetics, pharmacodynamics, and efficacy of a gene editing system targeting the human hydroxyacid oxidase 1 (HA01) gene in human patients having the primary hyperoxaluria type 1 (PHI) disease.I. Introduction of the HAOl-Targeting Gene Editing System

[0253] The HAO 1 -targeting gene editing system used in this example contains lipid nanoparticles (LNPs) complexed with two active ingredients: (1) a messenger RNA (SEQ ID NO: 23) encoding the CRISPR-associated nuclease Casl2i2 having the amino acid sequence of SEQ ID NO: 7; and (2) a synthetic gRNAhaving the nucleotide sequence of SEQ ID NO: 14, which specifically targets and disrupts the human HA01 gene. Details of the components of this HAO 1 -targeting gene editing system are provided below.

[0254] A. Projected Mechanism of Action

[0255] The HAO 1 -targeting gene editing system used herein acts by specifically altering the genome at the target locus to achieve a permanent loss of function of the HA01 gene. Durable disruption of the HA01 gene by the Casl2i2-mediated gene editing reduces glycolate oxidase (GO) activity in hepatocytes, which reduces the amount of available glyoxylate, a substrate for oxalate production. Targeted delivery to hepatocytes is achieved using lipid nanoparticles (LNPs) encapsulated with the Casl2i2-encoding mRNA and the HA01 gene-targeted gRNA. As the GO enzyme is upstream of the deficient AGT enzyme that causes PHI, the mechanism of action of the HAO 1 -targeting gene editing system is independent of the underlying AGXT gene mutation.

[0256] Nonclinical evaluation was performed to obtain relevant data for the benefit-risk assessment, for the selection of a clinical dose range, including a safe starting dose and dose escalation plan, and to manage overall safety of participants in the first-in-human clinical trial with the HAO 1 -targeting gene editing system disclosed herein.

[0257] In cultured primary human hepatocytes (PHH), the HAO 1 -targeting gene editing system demonstrated potent, on-target editing of HA01 gene with a conserved indel pattern across multiple donors. Genome- wide evaluations of HAO 1 -targeting gene editing system at supersaturating concentrations of HAO 1 -targeting gene editing system (1.5x the 90% effective concentration for on-target editing) in PHH yielded no detectable off-target editing in coding regions and the 3 off-target sites identified were within introns at editing levels close to the limit of detection. There were no genomic rearrangements.

[0258] A quantitative systems pharmacology (QSP) model was constructed to estimate a minimally efficacious starting dose for PHI patients. This model incorporated administration, distribution, and elimination of the HAO 1 -targeting gene editing system; specific distribution tothe liver; uptake by hepatocytes; HA01 target gene editing; and reduction in GO enzyme activity and was parameterized based on published data to calibrate the metabolic pathways of glyoxylate metabolism across animal species and metabolic states, including humans with PHI .

[0259] By standard allometric scaling, the 3 mg / kg no-observed-adverse-effect-level (NOAEL) in NHPs corresponds to an approximate human equivalent dose of 1 mg / kg based on a 75 -kg human. See Example 1 above.

[0260] B. Expected Clinical Benefits

[0261] The treatment of PHI using the HAO 1 -targeting gene editing system following the treatment conditions as disclosed herein would be expected to be safe and effective. The anticipated possible therapeutic and public health benefits following successful completion of this trial are that reduced production of oxalate by the liver will lead to less systemic oxalate, lower levels of UOx, and stabilization of renal function, with the goal of reducing disease burden and preventing disease progression. Based on nonclinical studies, the HAO 1 -targeting gene editing system disclosed herein is anticipated to specifically edit the HA01 gene in liver hepatocytes, thus preventing the metabolism of glycolate to glyoxylate, leading to increased plasma glycolate levels and reduced urinary oxalate levels.

[0262] C. Trial Objectives and Endpoints

[0263] Table 3. below outlines the trial objectives and endpoints.

[0264] Table 3. Objectives and Endpoints

[0265]

[0266]

[0267] > Abbreviations: BSA = body surface area; Casl2i2v2.1 = clustered regularly interspaced short palindromic repeats-associated protein 12i2; eGFR = estimated glomerular filtration rate; gRNA = guide ribonucleic acid; KDQOL-SF™ = Kidney Disease Quality of Life-Short Form; mRNA = messenger ribonucleic acid; PD = pharmacodynamics; PedsQL™ = Pediatric Quality of Life Inventory; PHI = primary hyperoxaluria type 1 ; PK = pharmacokinetics; SAEs = serious adverse events; TEAEs = treatment-emergent adverse events; UOx = urinary oxalate excretion.

[0268] II. Trial Design

[0269] A. Description of Trial Design

[0270] This open-label, dose-escalation, first-in-human Phase 1 / 2 trial is designed to determine the safety, tolerability, PK, and PD of the HAO 1 -targeting gene editing system in adult and pediatric participants aged >6 years to <64 years with PHI.

[0271] The trial is planned to consist of 2 Study Periods, having 2 parts and 4 cohorts (see Table 4 below). Study Period 1 has a Screening Period of up to 60 days, an Inpatient Stay of 5 days, and a Post-Dose Follow-Up until Month 24. Study Period 2 is a Long-Term Safety Follow-Up that lasts up to Year 15. Schedules of assessments for Period 1 and Period 2 are provided in Tables 5 and 6 below.

[0272] Part A will consist of 3 dose escalation cohorts in adults aged >18 to <64 years. In Part B, children aged >6 to <18 years will receive the HAO 1 -targeting gene editing system at the RD.

[0273] Table 4. Trial Parts and Cohorts

[0274] > <

[0275]

[0276] > <

[0277]

[0278] aDose cohorts may be expanded, or additional dose levels explored based on emerging clinical data to further characterize safety, tolerability, PK / PD, and early efficacy, with the aim of identifying an RD in adults that is well tolerated and efficacious in reducing urinary oxalate for patients with PHI.

[0279] bDose in mg / kg of the HAO 1 -targeting gene editing system refers to the amount of total RNA, (i.e., “1.0 mg / kg” is the total amount of guide RNA plus messenger RNA per kg of body weight).

[0280] cThe maximum dose of the HAO 1 -targeting gene editing system administered for a single participant will be limited to 75 mg (i.e., participants weighing 75 to 90 kg will receive a fixed 75 mg dose).

[0281] Abbreviations: PD = pharmacodynamics; PK = pharmacokinetics; RD = recommended dose; RNA = ribonucleic acid.

[0282] As the first-in-human investigation of the HAO 1 -targeting gene editing system, this trial includes an initial dose escalation phase in adults to establish a recommended dose (RD). The selected starting dose level of 0.4 mg / kg is estimated to be safe and minimally efficacious based on the totality of nonclinical toxicology and efficacy data, dose modeling, and extrapolated gene editing efficacy and safety from investigational agents in the same therapeutic class.Table 5. Schedule of Assessments - Study Period 1

[0283]

[0284] "

[0285]

[0286]

[0287]

[0288] 3If the participant reaches legal age for consent during trial participation, the participant should consent as an adult before any fiirther trial procedures may be performed.

[0289] bFull physical examination at Screening, Months 1, 12, 24, and at the Early Termination Visit. All other visits requiring physical examination will have a symptom-directed physical examination.

[0290] 5cSymptom-directed physical examination at Day -1 (pre-dose), Day 1 (post-dose), Days 2, 3, 4, 7, 14, and Months 2, 3, 6, and 18. Symptom-directed physical examinations can be done at the discretion of the Investigator throughout the trial.

[0291] dHeight to be collected at Screening, Days -1, Months 1, 2, 3, 6, 12, and 24 and at the Early Termination Visit for participants <18 years of age.

[0292] Participants >18 years of age will have height collected at Screening only. Height must be collected with shoes off.eWeight collected during Screening will be used to determine eligibility. Day - 1 weight will be used to determine pre- and post-medication dose. Shoes should be removed for weight collection.

[0293] fVital signs on the day of the infusion will be monitored at a minimum of pre-dose, every 30 minutes during the infusion, at the end of the infusion, at 1, 2, 4 and 6 hours after the infusion and then every 4 hours for the first 24 hours and as clinically indicated.

[0294] 5gTriplicate 12-lead ECG on Day 1 will be recorded at mid-infusion (approximately 1 hour post start of infusion), at the end of infusion (±30 minutes), and 4 hours post-dose (±30 minutes post end of infusion).

[0295] 11Pregnancy and childbirth status is only required for participants of childbearing potential.

[0296] 1Pregnancy test is only required for participants of childbearing potential. Serum pregnancy test to be performed at Screening and for confirmation of a positive urine pregnancy test. A urine pregnancy test can be performed at all other timepoints, but serum is also acceptable.

[0297] 101FSH testing is only required for female participants suspected of not being of childbearing potential.

[0298] kDay 1 blood sample for laboratory assessments should be collected >4 hours after the end of infusion; this sample can be collected at the same time as the sample for cytokines (6 hours [± 15 minutes] after the end of infusion).

[0299] 1Adverse events will be collected from the time of signing informed consent through the end of the Month 180 or Early Termination Visit.

[0300] mAll concurrent and prior medications taken within 30 days prior to signing informed consent will be recorded.

[0301] 15nLocal safety laboratory tests, to be performed for safety purposes, will include complete blood count, AST, ALT, PT and aPTT. The local laboratory reports may be requested by the Sponsor.

[0302] 0Complement (C3a, C5a and Bb) and cytokines (IL- ip, IL-2, IL-4, IL-6, IL-8, IL-10, TNFa, IFNy, and MCP-1) samples will be collected at 2 hours (±15 minutes) and 6 hours (±15 minutes) post-infusion.

[0303] 20pParticipants will receive prophylactic medications prior to administration of the HAO 1 -targeting gene editing system. For more details about premedications, see below.

[0304] qParticipants will receive a single dose of the HAO 1 -targeting gene editing system, administered via IV infusion over 2 hours. The duration of the infusion may be paused and / or extended in the event of an infusion -related reaction.. Participants should remain stationary for the duration of the infusion of the HAO 1 -targeting gene editing system.

[0305] 25rParticipants will receive a post-treatment dose of IV dexamethasone (adult dose 4 mg; pediatric dose 0.075 mg / kg up to maximum dose 4 mg) at 6 hours and 24 hours after completion of administration of the HAO 1 -targeting gene editing system.

[0306] seGFR will be based on the CKD-EPI 2021 equation for participants >18 years of age and both the updated Schwartz equation and the CKD-EPI 2021 equation for participants <18 years of age at Screening.

[0307] ‘ Participants will complete a diary during all 24-hour urine collections. Diary will be reviewed by site staff when urine collections are returned. 30uDuring the Screening Period, 3 separate 24-hour urine (±2 hours) collections will be completed. The mean of the first 2 valid 24-hour UOx will be used to determine eligibility. Eligibility urine samples will be collected for the assessment of urinary oxalate and will be analyzed at the central laboratory. For establishing baseline UOx, aliquots from all valid samples will be collected and analyzed by the specialist laboratory using a validated assay. Variation of estimated creatinine concentration between 2 valid samples cannot differ by more than 20%, otherwise the collection has to be repeated.

[0308] 35vThe Month 3, Month 6, and Month 12 urine samples will be collected (within 14 days prior to trial visit) for assessment of urinary oxalate, which will be analyzed centrally at a specialist laboratory using a validated assay.wSingle 24-hour urine to be collected within 7 days prior to trial visit. Urine samples will be collected for the assessment of urinary oxalate, which will be analyzed centrally at a specialist laboratory using a validated assay. If invalid, collection should be repeated.

[0309] xAll blood PD samples are to be collected at approximately the same time of day as the Day 1 pre-dose blood sample collection (±1 hour) during the Inpatient Stay. Blood samples will be collected for the assessment of plasma glycolate, which will be analyzed centrally using a validated assay.

[0310] 5ZPK samples will be collected pre-dose (within 60 minutes of infusion). During infusion, samples will be collected at 1 hour (±3 minutes), 2 hours (±3 minutes) and at the end of infusion (±3 minutes). Post-infusion samples will be collected at 30 minutes (±3 minutes), 4 hours (±15 minutes), 8 hours (±15 minutes), 24 hours (±30 minutes), and Day 4 (±60 minutes). Samples on Day 7 and beyond will be collected during the visit with other routine laboratory tests. All blood draws collected during infusion are to be done in a different limb than the infusion site (e.g., contralateral arm). A second IV line will be placed prior to the administration of the HAO 1 -targeting gene editing system to assist with blood collections during 10 the first 24 hours.

[0311] aaThe urine PK sample on Day 1 will be collected pre-dose followed by interval collection on Day 1 from 0-8 hours post start of infusion (SOI), 8- 26 hours post-SOI, 26-50 hours post-SOI, and 50-74 hours post-SOI. Times of collection as well as volume will be collected for each urination. Urine PK is for PL-A and CL-A only.

[0312] abScreening renal ultrasound to be completed as close to Day 1 as possible. Screening ultrasound can be completed up to Day 1 pre-dose. Results to 15 be analyzed by a central reader.

[0313] acRenal stone events will be collected using historical information from 12 months prior to Screening. Renal stone events will be collected during the trial from Screening until the end of the trial.

[0314] adParticipants who fail to qualify for the trial based on laboratory tests may be considered for rescreening at the discretion of the Investigator (limited to 2 screening attempts, first screening and 1 rescreening). Participants can have their screening extended for unexpected operational or logistical 20 delays. For further details regarding rescreening and for information about retesting and screening extension.

[0315] aeEligibility criteria must be confirmed before Day -1.

[0316] Prior to administration of prophylactic medication, there must be confirmation of a negative pregnancy test and collection of the local and central laboratory tests. Day -1 local laboratory results must be reviewed prior to the administration of the HAO 1 -targeting gene editing system.

[0317] agMonth 4 and Month 5 visits may be performed remotely. In this case, single 24-hour urine collections can be delivered to the site via courier from 25 their home or hotel, the 24-hour urine collection diary can be sent virtually, and the participant contacted via telephone to review and record adverse events, record concomitant medications, and collect renal stone events.

[0318] ahAn Early Termination Visit is only required for participants who prematurely discontinue the trial.

[0319] aiAssessments performed at the unscheduled visit are at the discretion of the Investigator.

[0320] Abbreviations: ALT = alanine aminotransferase; aPTT = activated partial thromboplastin time; AST = aspartate aminotransferase; CKD- 30 EPI = Chronic Kidney Disease Epidemiology Collaboration; DNA = deoxyribonucleic acid; ECG = electrocardiogram; eGFR = estimated glomerular filtration rate; FSH = follicle stimulating hormone; HBV = hepatitis B virus; HCV = hepatitis C virus; IFNy = interferon gamma; IL = interleukin; IV = intravenous; MCP-1 = monocyte chemoattractant protein-1; PD = pharmacodynamic; PHI = primary hyperoxaluria type 1; PK = pharmacokinetic; PT = prothrombin time; SOI = start of infusion; TNFa = tumor necrosis factor alpha; UOx = urinary oxalate excretion.Table 6: Schedule of Assessments - Study Period 2

[0321]

[0322]

[0323] aSymptom-directed physical examinations can be done at the discretion of the Investigator throughout the trial.

[0324] bHeight to be collected annually only for participants <18 years of age.

[0325] cPregnancy and childbirth status is only required for participants of childbearing potential. This assessment will be done at the trial clinic from Month 18 in Study Period 1 to Month 120 and via telephone call from Month 132 to Month 180.

[0326] dAdverse events will be collected from the time of signing informed consent through the end of the Month 180 or Early Termination Visit.

[0327] eLocal safety laboratory tests including liver function tests, serum chemistry, complete blood count, and coagulation will be performed by a local laboratory for safety purposes at the discretion of the participant’s regular care physician. The reports of any local safety testing done will be sent by the participant to the Investigator, who will alert the Sponsor in case of unusual findings.

[0328] feGFR will be based on the CKD-EPI 2021 equation for participants >18 years of age and both the updated Schwartz equation and the CKD-EPI 10 2021 equation for participants <18 years of age at Screening.

[0329] gSingle 24-hour urine to be collected within 7 days prior to trial visit. Urine samples will be collected for the assessment of urinary oxalate, which will be analyzed centrally at a specialist laboratory using a validated assay. If invalid, collection should be repeated.

[0330] 11Blood samples will be collected for the assessment of plasma glycolate, which will be analyzed centrally using a validated assay.

[0331] 1Renal ultrasound results to be analyzed by a central reader.

[0332] 151Renal stone events will be collected during the trial from Screening until the end of the trial.

[0333] kVisits from Year 5 to Year 10 can be aligned with standard of care visits.

[0334] 1Visits from Year 11 to Year 15 will be done via telephone call, to collect AEs, self-reported concomitant medications, and to administer the quality - of-life questionnaire.

[0335] mAn Early Termination Visit is only required for participants who prematurely discontinue the trial.

[0336] 20nAssessments performed at the unscheduled visit are at the discretion of the Investigator.

[0337] Abbreviations: AEs = adverse events; CKD-EPI = Chronic Kidney Disease Epidemiology Collaboration; eGFR = estimated glomerular filtration rate; PD = pharmacodynamic.NHP Good Laboratory Practice (GLP) toxicology studies were performed using the HAO 1 -targeting gene editing system since the HA01 target sequence is identical between cynomolgus monkey and humans. The highest dose of the HAO 1 -targeting gene editing system tested (3 mg / kg) was clinically well tolerated, with primary safety findings noted to be transient, asymptomatic, and resolving to baseline status by 2 weeks post dosing without accompanying cellular changes on histopathologic examination. An overall NOAEL of 3 mg / kg was established for the HAO 1 -targeting gene editing system, administered as a 1-hour infusion with the defined pre- and post-medication regimen.

[0338] By standard allometric scaling, the 3 mg / kg NOAEL in NHPs corresponds to an approximate human equivalent dose of 1 mg / kg based on a 75 -kg human. The lack of irreversible toxi cities, together with other toxicology findings that can be readily monitored (e.g., using standard laboratory markers of hepatotoxicity and coagulopathy), support the proposed clinical dose range starting at 0.4 mg / kg, with dose escalation to a high dose of 1.0 mg / kg. The maximum dose of the HAO 1 -targeting gene editing system administered for a single participant is limited to 75 mg (i.e., participants weighing 75 to 90 kg will receive a fixed 75 mg dose). This is based on, at least in part, the use of a 75 -kg human in allometric scaling from NHP data.

[0339] The RD is to be established based on the safety and preliminary efficacy (changes in urinary oxalate levels at Month 2 or Month 3) data from the adult cohorts in Part A, and is defined as a dose that is safe, tolerable, at or below the maximum tolerated dose (MTD) (if identified) and achieves clinically meaningful changes in urinary oxalate levels. Based on emerging data, additional participants may be added in any dose cohort, or additional dose levels may be explored in both pediatric and adult populations. Approximately 6 adult and 6 pediatric participants may be enrolled (for a total of approximately 23 [14 adult and 9 pediatric] participants) at the RD or other dosing levels to further characterize the safety and tolerability, and to provide additional safety information and / or initial determination of the efficacy of the HAO 1 -targeting gene editing system.

[0340] There is a sentinel dosing strategy in each cohort, consisting of a minimum of 28 -day dosing interval between the first and subsequent participants in the cohort. Selection of this dosing interval is based on nonclinical studies demonstrating that all acute laboratory abnormalities were transient and returned to baseline values by 2 weeks.

[0341] Patients with PHI, despite metabolic abnormalities, are anticipated to have healthy livers that effectively take up components of the HAO 1 -targeting gene editing system. This is incomparison to diseases such as heterozygous familial hypercholesterolemia, in which the haploinsufficiency of low-density lipoproteins receptors leads to impaired liver uptake of components of the HAO 1 -targeting gene editing system, potentially exposing extra-hepatic immune tissues to excess circulating LNPs at dose levels otherwise well tolerated in individuals with healthy livers.

[0342] The starting dose level of 0.4 mg / kg is expected to provide minimal efficacy that is observable in vivo. This dose level is modeled to provide a 25% reduction in UOx. Urinary oxalate has served in prior drug approvals for PHI as a surrogate endpoint for clinical efficacy. Altogether, the proposed starting dose level of 0.4 mg / kg balances expected safety, in vivo efficacy, and potential benefit to adult participants with PHI. Dose escalation proceeds to a middle (mid) dose of 0.7 mg / kg and a high dose of 1.0 mg / kg.

[0343] Initiation of the pediatric cohort (Cohort 4 disclosed below) is dependent on review of all available safety data from all treated participants and at least 2-3 months of urinary oxalate PD data post administration of the highest dose of the HAO 1 -targeting gene editing system. Based on available precedents and modeling, weight-based (mg / kg) dosing is expected to demonstrate similar safety and efficacy at a given weight-based (mg / kg) dose level in adults and pediatric participants (>6 years old).

[0344] For safety, the liver at > 6 years of age is both functionally mature and scales closely with weight as age increases (Coppoletta and Wolbach, 1933; Carpentieri et al., 1977; Small et al., 2017); thus, the effective hepatic uptake of LNP in children is expected to be similar to that in adults, without risk of excess circulating LNP exposure that may trigger additional toxicity. In the proposed Phase 1 / 2 trial described in this Example, additional safety measures are designed to further bridge safety from adult to pediatric participants in a stepwise manner, such as dosing a sentinel adolescent (>12 to <18 years old) participant to evaluate safety prior to enrollment of the full (>6 to <18 years) age range.

[0345] For PD, liver scaling with age and weight was incorporated into the QSP model to estimate the impact of age on efficacy of the HAO 1 -targeting gene editing system at a given dose. To achieve consistent levels of oxalate reduction in a 6-y ear-old, approximately 94% of the adult (21-year-old) dose is predicted to be needed, which is within the range of variability introduced by drug delivery and endpoint measurements. Additional dose levels may be explored in the pediatric population to adjust efficacy or to increase safety margins in response to emerging data.Dose-Limiting Toxicity

[0346] The dose-limiting toxicity (DLT) refers to treatment-related AEs or laboratory abnormalities that may be considered unacceptable in severity or duration and may prevent further dose escalation. The DLT observation period is 28 days. The proposed 28-day observation period was selected based on nonclinical studies indicating that by 4 weeks the active nuclease is cleared from the circulation and <1% of LNP lipid components are detected in the circulation. Once the 28-day DLT period has been completed for all participants in a cohort, and if no DLTs have been observed or safety concerns identified, then the sentinel participant in the next dose cohort can be enrolled.

[0347] AEs and laboratory abnormalities that are considered DLTs include:

[0348] • Any Grade 3 or higher AEs that is considered possibly, probably, or definitely related to the HAO 1 -targeting gene editing system except:

[0349] Grade 3 IRRs that fully resolve within 48 hours with standard supportive care and do not warrant elevation of level of care (e.g., admission to the hospital or to the intensive care unit).

[0350] • Any Grade 3 or higher laboratory abnormality that lasts >7 days and is considered possibly, probably, or definitely related to the HAO 1 -targeting gene editing system.

[0351] • Any Grade 4 laboratory abnormality that is considered possibly, probably, or definitely related to the HAO 1 -targeting gene editing system.

[0352] • Any other AEs or laboratory abnormality that based on reasonable clinical judgment, necessitates further characterization with respect to progression and reversibility.

[0353] Additional participants can be enrolled in a previous cohort or at an alternative lower dose. A dose reduction to an intermediate dose level between previously studied dose levels may be proposed if the administered dose is found to not be safe and well tolerated, or if the MTD has been exceeded. Dose escalation is to be considered complete after the RD has been identified or there is early termination of the cohorts.

[0354] Cohort 4 (pediatric participants) may start after:

[0355] • Available safety and PD data from the adult dose escalation cohorts have been reviewed for at least 28 days and no safety concerns have been identified, AND

[0356] • The RD has been determined.The first participant enrolled in Cohort 4 must be >12 to <18 years of age and follow the sentinel safety monitoring plan as described for the previous adult dose cohorts (Cohorts 1-3). If no concerns are identified, then the remaining participants in Cohort 4 can be >6 to <18 years of age. The remaining participant(s) in a cohort may be enrolled and treated simultaneously.

[0357] If a participant experiences a DLT, the time interval between participants within that cohort will be adjusted as shown in Table 7.

[0358] Table 7: Dosing Intervals

[0359]

[0360] Abbreviation: DLT = dose-limiting toxicity

[0361] B. Trial Procedures

[0362] Participants are to be screened for eligibility in the trial. The Screening Period lasts up to 60 days. Three urine collections are to be completed during the Screening Period to assess UOx and the mean UOx (oxalate concentration in 24-hour urine collection). The mean of the first 2 valid 24-hour urine collections evaluating UOx is to be used to determine eligibility.

[0363] Participants will be admitted to the trial site 1 day prior to administration of the HA01-targeting gene editing system. Participants receive oral dexamethasone 16-24 hours prior to administration of the HAO 1 -targeting gene editing system. Pre-medications (IV dexamethasone, IV Hi blocker, IV H2 blocker, IV acetaminophen / paracetamol) are to be administered 1-2 hours prior to administration of the HAO 1 -targeting gene editing system. Pre-medications are describedbelow. A second IV line is to be placed prior to the administration of the HAO 1 -targeting gene editing system to assist with blood collections during the first 24 hours.

[0364] A single dose of the HAO 1 -targeting gene editing system is to be administered via IV infusion on Day 1. A post-dose treatment of IV dexamethasone is to be administered 6 hours and 24 hours after completion of administration of the HAO 1 -targeting gene editing system.

[0365] Participants are observed as an inpatient for 4 days after administration of the HA01-targeting gene editing system. At the end of the Inpatient Stay, the participant may be discharged if clinically stable. Participants attend trial visits for 180 months after administration of the HA01-targeting gene editing system.

[0366] Toxicities are graded according to the CTCAE version 5.0 (or higher). PK assessments are performed at Baseline, during, and after the infusion. Routine sample collection for safety laboratory tests are conducted prior to dosing and throughout the trial. UOx is to be measured via 24-hour urine collections and renal ultrasound scans are performed to assess nephrocalcinosis. Antidrug antibodies (AD As) are measured to characterize the immunogenicity of the HA01-targeting gene editing system.

[0367] Cohort Progression and Stopping Rules

[0368] In this open-label trial, participants are allocated to the current dose cohort and receive the dose assigned to that cohort. Cohort progression and stopping rules are provided in Table 8 below.

[0369] Table 8. Cohort Progression and Stopping Rules

[0370] <

[0371] >

[0372]

[0373] >

[0374]

[0375] aAny event seen in a pediatric participant will be reviewed.

[0376] bReview of Part A cohorts for escalation to the next dose level will occur following at least 28 days of safety monitoring after the prior cohort has been dosed.

[0377] Abbreviations: ADR = adverse drug reaction SRB = safety review board

[0378] Criteria for Suspension of Enrollment

[0379] The occurrence of any of the following will result in immediate suspension of enrollment:

[0380] • Fatal or life-threatening AE that is considered possibly, probably, or definitely related to the HAO 1 -targeting gene editing system.

[0381] • Elevation in liver enzymes that is considered possibly, probably, or definitely related to the HAO 1 -targeting gene editing system and meets 1 of the following criteria:

[0382] o Elevation in aspartate aminotransferase (AST) or alanine aminotransferase (ALT) >5 x upper limit of normal (ULN) lasting for more than 4 weeks

[0383] • AST and ALT elevation >3 x ULN and total bilirubin >2 x ULN (Hy’s Law)

[0384] • Thrombosis, hemorrhage, and / or laboratory parameters consistent with disseminated intravascular coagulation and possibly, probably, or definitely related to the HA01- targeting gene editing system.

[0385] • Any other medically significant AE.

[0386] B. Rationale for Trial Design

[0387] The trial design aims to first establish safety and early efficacy in adult participants with PHI to establish an RD, followed by progression into pediatric participants with PHI. Theproposed inclusion of a pediatric population is justified by the expected safety of utilizing the same weight-based (mg / kg) dose from adults to the proposed pediatric age range and is further supported by the stepwise, data-driven approach to maximize the safety and prospect of direct benefit to pediatric participants.

[0388] 1. Weight-based dosing level (e.g. , mg / kg) is expected to be the same in the pediatric age range as in adult participants. Liver function and ability to maintain homeostasis are considered mature from age 6 (Grijalva and Vakili, 2013). In addition, liver size and volume as a percentage of body mass increases linearly from age 6 through adulthood (Coppoletta and Wolbach, 1933; Carpentieri et al., 1977; Small et al., 2017). Once a safe and well tolerated weight-based dose regimen for the HAO 1 -targeting gene editing system is determined in adults, it is expected that it can be safely applied to the proposed pediatric trial population of >6 years old.

[0389] 2. Dose escalation is to be performed in adult participants to determine an RD based on safety and early efficacy. The pediatric cohort is only to be initiated after an independent review of the totality of safety and PD data from all adult dose cohorts and the RD with an appropriate benefit / risk profile is established in adults. The selection of RD for Cohort 4 is to be further informed by the QSP modeling previously used to determine the starting dose in Cohort 1.

[0390] 3. A sentinel dosing strategy has been adopted for the pediatric population, such that the first pediatric participant is to be an adolescent >12 and <18 years of age, with safety supported by the nonclinical GLP toxicology study in NHPs. Clinical safety data generated in the sentinel adolescent participant and the totality of safety and efficacy data from adults provide further information to support dosing in participants >6 and <12 years old.

[0391] Performing dose escalation only in adult cohorts minimizes the risk that pediatric participants will either be exposed to a non-optimal efficacious dose or to a potentially toxic dose level. Initiating Cohort 4 at the RD maximizes the prospect of direct benefit in a disease with significant pediatric burden, and in a population where the advantages of a single-course, lifelong therapy may be the most clinically meaningful.C. Start and End of Trial

[0392] Trial participation is to last approximately 182 months, divided in 2 study periods. Study Period 1 includes Screening (up to 60 days), an Inpatient Stay of 5 days, and a Post-Dose Follow-Up of 24 months. Study Period 2 is a Long-Term Safety Follow-Up of 13 years (156 months).

[0393] Following completion of the Month 24 Visit, participants will enter Study Period 2, which is a long-term safety monitoring program created to comply with local regulation requirements / guidance for participants administered a gene editing therapy. During this period, assessments are to be done periodically to ensure participant long-term safety.

[0394] III. Trial Population

[0395] The trial population is intended to reflect the target indication of PHI, including, for example, adult and pediatric participants with PHI who have relatively preserved kidney function at eGFR >30 mL / min / 1.73 m2, elevated levels of urinary oxalate (mean 24-hour urinary oxalate >0.7 mmol / 24 hours / 1.73 m2, 1.5 x ULN), and no evidence of systemic oxalosis.

[0396] Participants with kidney function at an eGFR <30 mL / min / 1.73 m2and those on dialysis are not included, but may be examined in future trials, once the RD has been determined in participants with relatively preserved kidney function. The rationale for excluding participants with systemic oxalosis is that this condition limits rigorous assessment of the PD effect on urinary oxalate. As renal function worsens and eGFR declines, UOx decreases due to the inability of the kidneys to secrete oxalate into the urine, while plasma oxalate levels increase, leading to the development of systemic oxalosis. Measurement of a reduction in endogenous oxalate production from plasma or urine would be confounded by mobilization of oxalate from concentrated tissue deposits. Participants must also have AST, ALT, and total bilirubin laboratory values below the ULN for age.

[0397] Although PHI affects children as young as newborns, the proposed pediatric age range (>6 years to <18 years) for the trial is based on evaluating a disease-relevant demographic given the median age of PHI diagnosis is between 4 and 6 years of age, and including participants of an age that increases the probability of protocol adherence (e.g., valid 24-hour urine collections) for data robustness.

[0398] Inclusion Criteria

[0399] Human patients suitable for the clinical trial provided herein meet the following conditions:1. Confirmed diagnosis of PHI

[0400] 2. Age at time of signing the ICF / assent form:

[0401] a. Cohorts 1-3: >18 years to <64 years

[0402] b. Cohort 4: >6 years to <18 years (the first participant in Cohort 4 must be >12 to <18 years old)

[0403] 3. Documentation of PHI as determined by genetic analysis confirming pathogenic mutations in the alanine-glyoxylate aminotransferase (AGXT) gene

[0404] 4. Mean of 2 valid 24-hour UOx >0.7 mmol / 24 hours / 1.73 m2

[0405] 5. Able to follow directions and provide 24-hour urine collections

[0406] 6. If taking pyridoxine (vitamin B6) for the treatment of PHI, must have been on a stable regimen for at least 90 days prior to administration of the HAO 1 -targeting gene editing system, and be willing to remain on this stable regimen during Study Period 1

[0407] 7. Must meet the following laboratory entry criteria prior to enrollment in the trial: a. AST, ALT, total bilirubin <ULN for age. For participants with documented Gilbert’s syndrome, total bilirubin <2 x ULN

[0408] b. eGFR >30 mL / min / 1.73m2based on CKD-EPI 2021 equation for participants >18 years of age at Screening and both the Schwartz equation and the CKD-EPI 2021 equation for participants <18 years of age at Screening

[0409] c. Platelet count >100,000 / mm3

[0410] d. Normal PT, activated partial thromboplastin time (aPTT), international normalized ratio (INR), D-dimer, fibrinogen

[0411] 8. Participant must not have received any experimental agent for the treatment of PHI for at least 5 half-lives

[0412] 9. Participant must agree to not participate in another interventional trial during the Screening Period and for at least 6 months following administration of the HAO 1 -targeting gene editing system

[0413] 10. Weight <90 kg

[0414] 11. For female participants:a. If of childbearing potential, must agree to use at least 1 highly effective method of contraception from the time of signing the ICF through 12 months after dosing with the HAO 1 -targeting gene editing system; or

[0415] b. Be postmenopausal (defined as at least 1 year with no menses prior to Screening without an alternative medical cause); or

[0416] c. Be surgically sterile (e.g., post hysterectomy, bilateral salpingectomy, and bilateral oophorectomy) at least 1 month prior to Screening

[0417] 12. For male participants:

[0418] a. Must agree to use at least 1 highly effective method of contraception from the time of signing the ICF through 90 days after dosing with the HAO 1 -targeting gene editing system, and not donate sperm through 90 days after trial drug administration 13. Capable of providing signed informed consent. In case of participants who are below the legal age, informed consent must be obtained from the participant’s parent / LAR and the participant must provide assent per local and national requirements

[0419] 14. Must be willing to comply with the requirements of the trial

[0420] Exclusion Criteria

[0421] 1. Confirmed diagnosis of PH2 or PH3

[0422] 2. History of a liver, kidney, or combined liver / kidney transplant

[0423] 3. Currently on dialysis or anticipated requirement for dialysis within the initial 24 months of the trial (Study Period 1)

[0424] 4. Participant has previously used (within past 24 months) or is currently on an approved or investigational urinary oxalate lowering RNAi or siRNA therapy

[0425] 5. Medical history includes clinical evidence of extrarenal systemic oxalosis

[0426] 6. Known hypersensitivity to any LNP component or history of Grade 3 or higher AE following administration of any LNP requiring treatment or discontinuation of treatment, or any LNP treatment-related AE that may pose undue risk to the participant 7. History of liver cirrhosis

[0427] 8. Known or suspected systemic bacterial, viral, or fungal infection, or requirement of systemic anti-infectives within 14 days prior to trial drug administration9. History of active malignancy within 5 years prior to Screening except for adequately treated basal or squamous cell carcinoma of the skin, adequately treated cervical carcinoma in situ or adequately treated organ confined prostate cancer

[0428] 10. Female participants who are pregnant or breastfeeding (or are planning either during the first 12 months)

[0429] 11. History of alcohol or drug abuse within 3 years prior to Screening

[0430] 12. History of active hepatitis B, C or HIV infection; or positive hepatitis B surface antigen.

[0431] Participants who are hepatitis C virus (HCV) antibody positive must have negative HCVRNA

[0432] 13. Any condition or laboratory abnormality that in the opinion of the Investigator could pose undue risk, confound the ability to interpret trial results, or preclude compliance with the trial

[0433] 14. Anticipated survival <2 years

[0434] 15. Unwilling to comply with trial procedures including long-term safety follow-up

[0435] Lifestyle Considerations

[0436] The following lifestyle restrictions apply:

[0437] 1. Participants should refrain from consumption of high oxalate foods and vitamin C supplements for 1 week prior to all UOx assessments. High oxalate foods include but are not limited to chocolate, rhubarb, spinach, and beetroot.

[0438] 2. After being discharged from the Inpatient Stay, all participants are encouraged to stay within an estimated 2 hours of travel of the trial clinic until the Day 7 visit.

[0439] IV. HAO 1 -Targeting Gene Editing System and Concomitant Therapy

[0440] HAO 1 -Targeting Gene Editing System

[0441] The HAO 1 -targeting gene editing system for use in the instant example consists of LNP complexed with 2 active agents: a mRNA (SEQ ID NO: 23) encoding the CRISPR-associated protein 12i2 nuclease (SEQ ID NO: 7); and a synthetic gRNA (SEQ ID NO: 14), which specifically targets and disrupts the human HAO1 gene. The HAO 1 -targeting gene editing system is formulated as a liquid suspension for IV infusion as a single dose and is supplied as frozen liquid in glass vials.Dosing and Administration

[0442] Participants receive a single dose of the HAO 1 -targeting gene editing system, administered via IV infusion over 2 hours. The duration of the infusion may be paused and / or extended in the event of an infusion-related reaction. Infusion may be completed in 4 hours. Participants should remain stationary for the entire duration of the infusion.

[0443] Dose Escalation

[0444] Dose escalation is to be performed in adult participants (Cohorts 1 -3), at 3 planned dose levels. The starting dose is projected to be safe based on nonclinical GLP toxicology studies and minimally efficacious based on dose modeling to achieve a decrease in urinary oxalate concentration. The mid and high doses are selected to provide greater efficacy than the preceding lower dose while still remaining at or below the maximum safe dose extrapolated from nonclinical data.

[0445] Recommended Dose (RD) Selection

[0446] The RD is a dose that demonstrates safety, tolerability, and achieves the targeted reduction in 24-hour -UOx at Month 2 or Month 3 from the baseline value (defined as the average of the first 2 valid 24— hour urine collections during the Screening Period) based on totality of data from a cohort. The RD is to be at or below the MTD, the highest dose that has been found to be safe and well tolerated.

[0447] Treatment of Overdose

[0448] There is no antidote to the HAO 1 -targeting gene editing system disclosed herein. In the event of an overdose, the participant should receive supportive care and monitoring, as applicable. The Medical Monitor should be notified. The amount of the excess dose and duration of the overdose should be documented in the eCRF.

[0449] Preparation, Handling, Storage, and Accountability

[0450] The HAO 1 -targeting gene editing system is to be supplied as frozen liquid in glass vials and should be stored at -80 ± 10°C in a secure and locked location until ready to be thawed and combined into a saline bag according to participant weight for IV infusion. Dose preparation will require multiple vials of the gene editing system for all participants.Treatment Period

[0451] Participants will receive 1 dose of the HAO 1 -targeting gene editing system and be followed for 180 months (15 years).

[0452] Infusion-Related Reactions

[0453] The infusion rate is to be slowed or stopped in the event of an infusion-related reaction.

[0454] Prevention of Infusion-Related Reactions

[0455] For infusion-related reactions, drugs used to faciliate symptom resolution and resumption of the administration of the gene editing system include acetominophen / paracetamol, non-steroidal anti-inflammatory drugs, and diphenhyramine / Hi antihistamines (per package insert).

[0456] For severe infusion-related reactions, participants may receive additional corticosteroids. In severe cases, there may be hemodynamic instability and organ dysfunction, and require management with IV fluids and supplemental oxygen. Partipants should be closely monitored until resolution of the symptoms, including vital sign assessment every 4 hours and symptom evaluation twice a day. Participants with severe infusion-related reactions should have laboratory assessments including daily complete chemistry panel, lactate dehydrogenase (LDH), liver transaminases, bilirubin, coagulation panel (PT, aPTT, fibrinogen, D-dimer), C-reactive protein and ferritin. For persistent symptoms beyond 12-18 hours, participants may receive additional corticosteroids and tocilizumab at 24 hours. For persistent symptoms beyond 24 hours, additional IL-6 or IL-1 directed therapy, such as siltuximab or anakinra, should be considered.

[0457] Prior and Concomitant Medications

[0458] Recording of Prior and Concomitant Medications

[0459] All prescription and nonprescription medications (e.g., over-the-counter [OTC] medications and herbal supplements) that participants report taking during the 30 calendar days prior to signing the ICF (and assent, if applicable) should be recorded as prior medications.

[0460] All medications and therapies used after administration of the HAO 1 -targeting gene editing system should be recorded as concomitant medications.Prohibited Concomitant Medications

[0461] The following medications and therapies are prohibited starting from Screening until the final follow-up visit:

[0462] • RNAi or siRNA therapies

[0463] Permitted Concomitant Medications

[0464] The following medications and therapies are permitted starting from Screening until the final follow-up visit:

[0465] • Standard of care PHI treatments according to local / institutional guidelines including hyperhydration and crystallization inhibitors, with the exception of RNAi and siRNA therapies. Standard of care treatment may be adjusted beginning at Month 12.

[0466] • Pyridoxine supplementation (vitamin B6).

[0467] • Vitamin C supplements are permitted except during the week immediately prior to each urinary oxalate assessment.

[0468] • Medications used to prevent and treat infusion-related reactions and hyperglycemia, as described herein.

[0469] Prophylactic Medications

[0470] Prophylactic medications are administered to minimize the risk of infusion-related reactions according to Table 9 below.

[0471] Hyperglycemia may occur with dexamethasone prophylaxis; monitoring is recommended.

[0472] Table 9: Prophylactic Medications

[0473]

[0474] < >

[0475]

[0476] ote: Oral doses can be provided as a tablet or a liquid.

[0477] V. Trial Assessments and Procedures

[0478] The schedules of assessment are provided in Tables 4 and 5 above. The Screening visit is to occur within 60 calendar days prior to administration of the HAO 1 -targeting gene editing system. Each participant is to be screened to ensure eligibility for the trial. The Screening visit may be split over several days to complete all tests and assessments.

[0479] Demographic and Baseline Characteristics

[0480] Demographic and baseline characteristics to be recorded include: (a) age or year of birth; (b) weight and height; (c) date of PHI diagnosis, (d) other relevant medical history, and (e) prior medications.

[0481] Safety Assessments and Procedures

[0482] (1) Physical Examination

[0483] When required, a full physical examination will include examination of general appearance, skin, neck (including thyroid), eyes, ears, nose, throat, heart, lungs, abdomen, lymph nodes, extremities, and nervous system. At other time points as noted in Tables 4 and 5, a symptom-directed examination will be conducted that is focused on any changes since the previous examination. A symptom-directed physical examination can be done at the discretion of the Investigator throughout the trial.

[0484] (2) Height and Body WeightHeight without shoes (in centimeters) is to be recorded at Screening for all participants. During Study Period 1, pediatric participants have their height measured at the time points indicated in Table 4 and at Month 24 or Early Termination Visit. During Study Period 2, pediatric participants continue to have their height measured according to the schedule of assessment shown in Tables 4 and 5 until they reach 18 years.

[0485] Body weight (in kilograms) without shoes will be recorded for all participants at the time points indicated in the schedule of assessment shown in Tables 4 and 5.

[0486] (3) Vital Signs

[0487] Vital signs include infrared body temperature, respiratory rate, seated radial pulse rates, and seated systolic and diastolic blood pressures (BPs). Seated recordings are to be made after the participant has rested comfortably in the seated position for at least 3 minutes with their feet squarely on the floor and arm relaxed, bent at the elbow.

[0488] (4) Electrocardiogram

[0489] Triplicate 12-lead ECGs will be measured approximately 5 minutes apart for a total of roughly 15 minutes. Recordings are obtained after the participant has rested comfortably in the supine position for approximately 10 minutes. Participants should remain supine between ECGs. ECGs should be recorded at approximately the same time of day during the inpatient stay. ECGs are also to be collected at onset of chest pain or other cardiac symptoms. Additional ECGs may be collected.

[0490] The electrophysiological parameters assessed include, but are not limited to, rhythm, ventricular rate, PR interval, QRS duration, QT interval, ST and T waves, and Fridericia-corrected QT interval (QTcF).

[0491] (5) Clinical Laboratory Tests

[0492] The following clinical laboratory tests are to be performed centrally as indicated in the schedule of assessment shown in Tables 4 and 5:

[0493] • Hematology: hemoglobin, hematocrit, red blood cell count, white blood cell count (with differential), and platelet count.

[0494] • Serum Chemistry: albumin, albumin / globulin ratio, ALT, alkaline phosphatase, AST, bilirubin-direct, bilirubin-indirect, total bilirubin, blood urea nitrogen (BUN), BUN / creatinine ratio, calcium, cholesterol, chloride, creatine kinase,creatinine, gamma-glutamyl transpeptidase, ferritin, globulin, glucose, LDH, phosphate, potassium, total protein, sodium, triglycerides, and uric acid.

[0495] • Coagulation: D-dimer, fibrinogen, INR, PT, and aPTT.

[0496] • Urinalysis: pH of freshly voided specimen, specific gravity, protein, glucose, ketones, blood, and microscopic examination of the sediment.

[0497] • PH 1 AGXT Genotyping', if a valid historical sample is not available, a DNA sample will be collected at Screening for confirmation of PH1 / AGAT mutation.

[0498] • PK, PD, and Immunogenicity', plasma concentrations of the lipid components in the gene editing system (PL- A, CL- A), Casl2i2 mRNA, and gRNA, urine concentrations of the lipid components (PL-A and CL-A), 24-hour UOx, plasma glycolate, eGFR, AD As, anti-Casl 2i2.

[0499] • Other: HIV, HBV and HCV (including HCV RNA) serologies, C-reactive protein, complement (C3a, C5a and Bb), cytokines (IL-ip, IL-2, IL-4, IL-6, IL- 8, IL-10, TNFa, IFNy, and MCP-1), FSH for female participants suspected of not being of childbearing potential, serum pregnancy test for participants of childbearing potential (mandatory at Screening and for confirmation of local urine testing. Serum or urine can be used at other timepoints). Laboratory samples are analyzed by a central laboratory to ensure consistent interpretation of results. Local laboratory tests are to be performed on Day-1 for safety monitoring. Tests to be performed locally are complete blood count, AST, ALT, PT, and aPTT. Serum tryptase and plasma histamine will alsobe performed locally if anaphylaxis is suspected. Non-protocol specified laboratory results that are needed to manage a participant should be reported as an SAE or AE if they are deemed clinically significant.

[0500] Adverse Events and Serious Adverse Events

[0501] (1) Definitions and Criteria

[0502] Adverse Events

[0503] Per ICH E2A (International Council for Harmonization, 1994), an AE is “any untoward medical occurrence in a patient or clinical investigation patient administered a pharmaceutical product and which does not necessarily have to have a causal relationship with this treatment. AnAE can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding, for example), symptom, or disease temporally associated with the use of a medicinal product, whether or not considered related to the medicinal product.”

[0504] For the purposes of this clinical trial, renal stone events, with the exception of those events leading to hospitalization for more than 24 hours, will not be considered AEs since these events are expected in patients with PHI.

[0505] Medical interventions such as surgeries, diagnostic procedures, and therapeutic procedures are not AEs, but the action taken to treat the medical condition. They should be recorded as treatment(s) of the AEs.

[0506] Serious Adverse Events

[0507] Per ICH E2A (International Council for Harmonization, 1994), an SAE or serious suspected adverse reaction is “any untoward medical occurrence or effect that at any dose results in death, is life-threatening, requires hospitalization or prolongation of existing hospitalization, results in persistent or significant disability or incapacity, or is a congenital anomaly or birth defect”. Important medical events that may not result in death, be life-threatening, or require hospitalization, may be considered an SAE when, based upon appropriate medical judgment, they may jeopardize the participant and may require medical or surgical intervention to prevent one of the outcomes listed in this definition. Examples of such events are:

[0508] • Intensive treatment in an emergency room or at home for allergic bronchospasm • Blood dyscrasias or convulsions that do not result in inpatient hospitalization For deaths, the underlying or immediate cause of death should always be reported as an SAE.

[0509] An AE is life-threatening if the participant was at immediate risk of death from the event; it does not refer to an event that hypothetically might have caused death if it were more serious. For example, trial drug-induced hepatitis that resolved without evidence of hepatic failure would not be considered life-threatening even though trial drug-induced hepatitis can be fatal.

[0510] All AEs requiring hospitalization greater than 24 hours should be considered SAEs. Hospitalization for elective surgery or routine clinical procedures that are not the result of AEs (e.g., elective surgery for a pre-existing condition that has not worsened) need not be consideredAEs or SAEs. If anything untoward is reported during the procedure, that occurrence must be reported as an AE, either ‘serious’ or ‘nonserious’ according to the criteria outlined above.

[0511] An AE is incapacitating or disabling if the experience results in a substantial and / or permanent disruption of the participant's ability to carry out normal life functions.

[0512] Assessment of Severity

[0513] Each AE is to be classified according to the CTCAE criteria v5.0 (CTCAE v5.0, 2017) as shown in Table 10 below:

[0514] Table 10. Severity of Adverse Events

[0515]

[0516] Severity versus Seriousness: Severity is used to describe the intensity of a specific event while the event itself may be of relatively minor medical significance (such as severe headache). Severity is not the same as “seriousness”, which is based on parti cipant / event outcome at the time of the event. When changes in the severity of an AE occur more frequently than once a day, the maximum severity for the experience should be noted. If the severity category changes over several days, those changes should be recorded as separate AEs (with distinct onset dates).Adverse Events of Special Interest

[0517] The following events and / or laboratory findings are designated as adverse events of special interest (AESIs) based on the predicted pharmacology, nonclinical safety profile, possible extrahepatic targeting of the HAO 1 -targeting gene editing system, adverse reactions observed in systemically delivered LNP products, and / or adverse reactions from prophylactic medications.

[0518] • Moderate to severe infusion-related reactions (CTCAE >Grade 2)

[0519] • Acute liver injury evidenced by CTCAE >Grade 2 elevations in AST, ALT, total bilirubin, or clinically relevant symptoms or signs of liver injury

[0520] • Abnormal coagulation findings as evidenced by clinically relevant abnormal bleeding, thrombotic or hemorrhagic events, CTCAE >Grade 2 abnormal blood test results (e.g., increased PT >1.5 x ULN)

[0521] • Muscle injury evidenced by CTCAE >Grade 2 elevations in creatine kinase >2.5x ULN

[0522] • AEs attributed to the spleen (e.g., systemic inflammation or sepsis) Assessment of Relationship

[0523] The relationship to the HAO 1 -targeting gene editing system for each AE, based on the following criteria:

[0524] • Not related: No causal relationship exists between the administration of the gene editing system and the AE but an obvious alternative cause exists, e.g., the participant’s underlying medical condition or concomitant therapy • Possibly related: A connection with the administration of the gene editing system appears unlikely but cannot be ruled out with certainty. An AE may be considered possibly related if or when it meets 2 of the following criteria: (1) it follows a reasonable temporal sequence from administration of the trial drug; (2) it could not readily have been produced by the participant’s clinical state, environmental or toxic factors, or other modes of therapy administered to the participant; or (3) it follows a known pattern of response to the trial drug

[0525] • Probably related: A connection with the administration of the gene editing system appears unlikely but cannot be ruled out with certainty. An AE may be considered probably related if it follows a reasonable temporal sequence from administrationof the gene editing system or the prophylactic medications and if it is a lesser known or suspected effect of the gene editing system or the prophylactic medications

[0526] • Related: There is a reasonable / plausible possibility that the AE may have been caused by the gene editing system

[0527] When assessing the relationship to the trial drug, the following criteria will be considered:

[0528] • Known class effect.

[0529] • Biological plausibility.

[0530] • Lack of alternative explanation (e.g., a concomitant drug or disease).

[0531] Action Taken for Event

[0532] The action(s) taken as a result of the AE should be selected from 1 of the categories listed below and recorded:

[0533] • None (i.e., no treatment was required).

[0534] • Therapy(ies) required (i.e., prescription and / or OTC medication was required to treat the AE).

[0535] • Hospitalization or prolongation of hospitalization required (i.e., hospitalization was required or prolonged because of the AE, whether medication was required).

[0536] • Other.

[0537] Pharmacodynamic Assessments and Procedures

[0538] UOx is to be determined from 24-hour urine collections. During the Screening Period, 3 separate 24-hour urine collections are to be completed. The mean UOx in the first 2 valid 24-hour urine collections are used to determine eligibility. For establishing baseline UOx, aliquots from all valid screening samples will be collected and analyzed by the specialist laboratory using a validated assay. Participants also provide 3 separate 24-hour urine collections and >2 of these collections must be valid at the Month 3, Month 6, and Month 12 Visits. Single 24-hour urine collections will be obtained at the other timepoints as specified in the schedule of assessments provided in Tables 4 and 5. The 24-hour urine collections can be taken by the participant to the trial site or sent via courier. eGFR will be calculated based on the CKD-EPI 2021 equation for participants >18 years of age and both the updated Schwartz equation and the CKD-EPI 2021 equation for participants <18 years of age at Screening.PD blood samples are collected at approximately the same time of day corresponding to the pre-dose collection (±1 hour) during the inpatient stay. Blood samples are collected and will be analyzed centrally for plasma glycolate using a validated assay.

[0539] Validity Criteria for 24-hour Urine Collections

[0540] A 24-hour urine collection is considered valid if all the following criteria are met:

[0541] • The collection is between 22-26 hours in duration between the initial discarded void and the last void or attempted void

[0542] • No voids are missed between the start and end time of the collection as indicated by the participant’s 24-hour urine collection diary

[0543] • The 24-hour creatinine content is at least 10 mg / kg as assessed by the central laboratory

[0544] • During Screening or when multiple 24-hour urine collections are required at a given timepoint, variation in 24-hour urinary creatinine values cannot be more than 20% between 2 of the samples

[0545] Pharmacokinetic Assessments and Procedures

[0546] Details of the procedures to be followed for sample collection, storage, and shipment are to be documented in a separate Laboratory Manual.

[0547] The PK of the lipid components, the Casl2i2 mRNA, and the gRNA in plasma and the lipid components in urine are to be assessed.

[0548] Genetics

[0549] If a valid historical sample is not available, a DNA sample is to be collected at Screening for confirmation of PHI / A GXT mutation.

[0550] Immunogenicity Assessments

[0551] AD As to the gene editing system and anti-Casl 2i2 protein antibodies are to be evaluated in plasma.

[0552] Exploratory Assessments and Procedures

[0553] Renal Ultrasound Scans and Renal Stone Events

[0554] Nephrocalcinosis is to be assessed by renal ultrasound scans. Changes in nephrocalcinosis grade will be assessed using a semi-quantitative scale (Dick et al., 1999). Renal stone events areassessed from 12 months prior to Screening until the end of the trial. Renal stone events prior to Screening are collected using historical information. Events include visits to a healthcare provider for renal stones, medications for renal colic, stone passage, or macroscopic hematuria due to renal stones.

[0555] Quality of Life Questionnaire

[0556] Pediatric Quality of Life Inventory (PedsQL)™

[0557] The Pediatric Quality of Life Inventory (PedsQL™) measures quality of life (QoL) in healthy children and adolescents with acute and chronic health conditions (Varni et al., 1999; Varni et al., 2001). The PedsQL™ Generic Core Scales take less than approximately 4 minutes to complete, are developmentally appropriate, and are translated into multiple languages. The Generic Core Scales contain 23 items that measure core health domains, including physical, emotional, school and social functioning. Scoring is summarized as Total Scale Score, Physical Health Summary Score, and Psychosocial Health Summary Score. The PedsQL™ is completed by the participants or proxy (such as the participant’s parent / LAR, should the participant be unable to self-report). The measure is valid for pediatric and adolescent participants who are <18 years of age at the time of signing the informed consent / assent.

[0558] Kidney Disease Quality of Life-Short Form (KDQOL-SF)™

[0559] The Kidney Disease Quality of Life-Short Lorm (KDQOL-SL)™ is a self-reported measure developed for individuals with kidney disease and those on dialysis (Hays et al., RAND Corporation, Version 1.3, 1997). It includes 43 kidney disease-targeted items, such as the effects of the disease on activities of daily living, work status, and social interaction, and 36 items that provide a measure of physical and mental health, and 1 overall health rating item ranging from 0 (“worst possible health”) to 10 (“best possible health”). The 80 items take about 16 minutes to complete.

[0560] VI. Statistical Considerations

[0561] A detailed Statistical Analysis Plan (SAP) for Study Period 1 is to be finalized and signed before the first patient is dosed.Sample Size

[0562] Due to the rare nature of the clinical indication targeted, the sample size in this trial is not based on any formal statistical assessment but was deemed sufficient to meet the trial objectives.

[0563] Analysis Sets

[0564] Safety Analysis Set: All enrolled participants who receive an infusion of the gene editing system. The Safety Analysis Set serves as the analysis population for all safety analyses.

[0565] DLT Evaluable Analysis Set: All enrolled participants who receive an infusion of the gene editing system and are followed for safety over the entire DLT observation period or experience DLT within 28 days after administration of the gene editing system.

[0566] PK Analysis Set: All enrolled participants who receive an infusion of the gene editing system and have at least 1 post-dose sample for PK parameters and who have evaluable PK data (blood or urine).

[0567] Modified Intention to Treat (mITT) Analysis Set: All enrolled participants who receive an infusion of the gene editing system, have at least 1 post-dose PD measurement (blood or urine) or 1 post-dose exploratory endpoint value, and who do not have any major protocol deviations.

[0568] Immunogenicity Analysis Set: All enrolled participants with available ADA data and no protocol deviations with a relevant impact on ADA data.

[0569] Safety Analyses

[0570] Descriptive statistics (such as means, medians, standard deviations, and ranges) are presented for continuous data, and frequencies and percentages are presented for discrete / categorical variables.

[0571] Adverse Events

[0572] All AEs are coded using version 27.0 or higher of the Medical Dictionary for Regulatory Activities (MedDRA). The incidence of TEAEs, SAEs and adverse drug reactions (ADRs) are summarized by cohort and overall.

[0573] A TEAE is defined as any AE that has an onset during or after administration of the gene editing system, or any pre-existing condition that has worsened during or after administration of the gene editing system. An ADR is an AE that is considered possibly, probably, or definitely related to administration of the gene editing system. The incidence of TEAEs and treatment-related AEs is also to be summarized by maximum severity and relationship to the gene editing systemby MedDRA primary system organ class and preferred term. The summary includes the total number and percentage of participants reporting a particular event.

[0574] A summary of AEs of CTCAE Grade 3 or higher is provided using MedDRA version 27.0 or higher. The incidence of infusion-related reactions and injection site reactions are also be summarized by MedDRA primary system organ class and preferred term.

[0575] Dose-Limiting Toxicity Adverse Events

[0576] DLTs are summarized for the DLT Evaluable Analysis Set and listed.

[0577] Electrocardiogram

[0578] A list of participants with abnormal 12-lead ECG parameters are presented. Baseline and change from Baseline in ECG parameters are summarized with descriptive statistics at each postBaseline time point.

[0579] Vital Signs

[0580] Descriptive statistics for baseline values and change from Baseline in vital sign parameters will be summarized at each post-Baseline time point.

[0581] Physical Examination

[0582] Full physical examination data is to be summarized with descriptive statistics by cohort and visit. All physical examination data (full and symptom-directed) will be listed.

[0583] Clinical Laboratory Tests

[0584] Mean changes from Baseline at each post-Baseline time point for each laboratory parameter will be presented for centrally reported values. Clinical laboratory values are graded according to the CTCAE v5.0 or higher for applicable tests. In addition, each reading is classified as below, within, or above normal range, based on ranges supplied by the laboratory used. Furthermore, any confirmed elevation in liver enzymes and / or alteration of coagulation parameters, identified as per Sections “Criteria for Suspension of Enrollment” and “Cohort Progression and Stopping Rules”, are to be summarized and listed. Shift tables for the baseline and follow-up measurements are presented.

[0585] Analysis Supporting Secondary Objectives

[0586] Pharmacodynamic AnalysesAll PD analyses will be based on the mITT Analysis Set. PD parameters to be assessed include the following:

[0587] • Percent change in 24-hour UOx from baseline to Month 6 (the mean of Month 3 and Month 6 UOx measurements)

[0588] • Absolute change in UOx corrected for BSA

[0589] • Percent change in plasma glycolate from Baseline to Month 6 (the mean of Month 3 and Month 6 plasma glycolate measurements)

[0590] • Changes in eGFR from Baseline to Month 12 and Month 24.

[0591] Each parameter is summarized by dose using descriptive statistics. The time course for urinary oxalate excreted is displayed graphically by dose. PD parameters versus dose are plotted. The annualized rate of change in eGFR is calculated as a slope of a simple regression over time.

[0592] It is noted that reduction in 24 hour urinary oxalate was observed at the low dose.

[0593] Pharmacokinetics

[0594] To assess PK, plasma concentration and time data will be tabulated and plotted by dose level. The PK of the HAO 1 -targeting gene editing system is summarized by estimating area under the curve (AUC) to the last measurable concentration (AUChst), AUC to infinity (AUCinf), maximum plasma concentration (Cmax), time taken to reach Cmax (Tmax), half-life (ti / 2), clearance (CL), and volume of distribution (Vd) as appropriate for the data collected for 4 components of the gene editing system: the two lipid components, the Casl2i2 mRNA, and the gRNA.

[0595] Urine concentration data is tabulated and plotted for the lipid components, if measurable. If available, the parameters are presented by dose cohort and descriptive summary statistics are to be generated. Otherwise, the measurable data is to be listed.

[0596] The relationship between PK parameters and certain safety, activity, and PD variables may be explored via modeling.

[0597] Immunogenicity

[0598] AD As to the gene editing system and anti-Casl 2i2 protein antibodies are assessed by dose cohort using descriptive statistics for each trial part. All ADA data is to be listed for each participant.

[0599] Analysis of Exploratory ObjectivesNephrocalcinosis

[0600] Changes in nephrocalcinosis grade are assessed via ultrasound and reported using descriptive statistics (Dick et al., 1999) for baseline and change from baseline values at Month 12 and 24. The analysis is to be done on the mITT Analysis Set.

[0601] Renal Stone Events

[0602] Renal stone events will be assessed from 12 months prior to Screening until the end of the trial for each cohort separately. Renal stone events prior to Screening will be collected using historical information. Events will include visits to a healthcare provider for renal stones, medications for renal colic, stone passage, or macroscopic hematuria due to renal stones.

[0603] The renal stone event incidence rate will be calculated as the number of renal stone events divided by participant exposure time (time since administration of the gene editing system) and multiplied by 1,000. The analysis will be done on the Safety Analysis Set.

[0604] Other Analyses

[0605] Patient-reported QoL data is to be summarized as the observed value at baseline and change from baseline through the end of the trial. The analysis will be done on mITT Analysis Set.

[0606] OTHER EMBODIMENTS

[0607] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0608] From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

[0609] EQUIVALENTS

[0610] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scopeof the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0611] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0612] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0613] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0614] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionallyincluding elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0615] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0616] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to ± 20 %, preferably up to ± 10 %, more preferably up to ± 5 %, and more preferably still up to ± 1 % of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” is implicit and in this context means within an acceptable error range for the particular value. In some embodiments, the hinge domain is a hinge domain of a naturally occurring protein.

[0617] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at leastone of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0618] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Claims

1. WHAT IS CLAIMED IS:

1. A method for treating primary hyperoxaluria 1 (PHI), comprising administering to a subject in need thereof an effective amount of a gene editing composition, which comprises:(a) a messenger RNA (mRNA) encoding a Casl2i2 polypeptide, which comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1, wherein the Casl2i2 polypeptide comprises at least amino acid substitutions D581R, I926R, and V1030G relative to SEQ ID NO: 1;(b) a guide RNA (gRNA) comprising a spacer sequence, which is specific to a target sequence of 5’-CAAAGTCTATATATGACTAT-3’ (SEQ ID NO: 10) in the HAO1 gene; and(c) and lipid excipients, which comprise a cationic lipid, a zwitterionic phospholipid, cholesterol, a pegylated lipid, or a combination thereof;wherein the mRNA and gRNA are associated with the lipid excipients, and optionally wherein the lipid excipients form lipid nanoparticles (LNPs) and the mRNA and gRNA are attached to or encapsulated by the LNPs; andwherein the gene editing composition is administered to the subject at an amount of total RNA of about 0.4 mg / kg to about 1 mg / kg.

2. The method of claim 1, wherein the amount of total RNA administered to the subject does not exceed 75 mg.

3. The method of claim 1 or claim 2, wherein the amount of total RNA administered to the subject is about 0.4 mg / kg, about 0.7 mg / kg, or about 1.0 mg / kg.

4. The method of any one of claims 1-3, wherein the Casl2i2 polypeptide comprises: (a) D581R, I926R, and V1030G relative to SEQ ID NO: 1; or(b) D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G relative to SEQ ID NO: 1.

5. The method of claim 4, wherein the Casl2i2 polypeptide comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

6. The method of any one of claims 1-5, wherein the Casl2i2 polypeptide further comprises a first nuclear localization signal (NLS) at the N-terminus, a second nuclear localization signal (NLS) at the C-terminus, or both, of the Casl2i2 polypeptide; optionally wherein the first NLS and the second NLS are identical.

7. The method of claim 6, wherein the first NLS and / or the second NLS is set forth as KRPAATKKAGQAKKKK (SEQ ID NO: 5).

8. The method of claim 6 or claim 7, wherein the Casl2i2 polypeptide further comprises a peptide linker between the NLS and the rest of the polypeptide; optionally wherein the peptide linker is set forth as SAGGGGSGGGGSGGGGSG (SEQ ID NO: 6).

9. The method of any one of claims 6-8, wherein the Casl2i2 polypeptide comprises the first NLS at the N-terminus and the second NLS at the C-terminus, and wherein the Casl2i2 polypeptide comprises the peptide linker connecting each of the first NLS and the second NLS to the rest of the polypeptide.

10. The method of claim 9, wherein the Casl2i2 polypeptide comprises the amino acid sequence of SEQ ID NO: 7.

11. The method of claim 10, wherein the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 8 and encodes the amino acid sequence of SEQ ID NO: 7.

12. The method of any one of claims 1-11, wherein the mRNA comprises a 5’ cap moiety and a 3’ polyadenylation tail.

13. The method of any one of claims 1-12, wherein the mRNA comprises one or more modified nucleotides, which comprise pseudouridine; optionally wherein at least 70% of the uridine positions in the mRNA contain pseudouridines.

14. The method of claim 1, wherein the mRNA comprises the nucleotide sequence of SEQ ID NO: 23; optionally wherein the mRNA comprises a 5’ cap moiety, and pseudouridine residues at all uridine positions in the mRNA.

15. The method of any one of claims 1-14, wherein the gRNA comprises the spacer sequence set forth as 5’-CAAAGUCUAUAUAUGACUAU-3’ (SEQ ID NO: 11).

16. The method of any one of claims 1-15, wherein the gRNA further comprises a direct repeat sequence, which optionally is 23-26 nucleotide in length.

17. The method of claim 16, wherein the direct repeat sequence is 5’-AGAAAUCCGUCUUUCAUUGACGG-3’ (SEQ ID NO: 12).

18. The method of any one of claims 1-17, wherein the gRNA comprises the nucleotide sequence of 5’-AGAAAUCCGUCUUUCAUUGACGGCAAAGUCUAUAUAUGACUAU-3’ (SEQ ID NO: 13).

19. The method of any one of claims 1-18, wherein the gRNA comprises one or more modifications, which optionally comprises phosphorothioate linkage and 2’-O-methylation; optionally wherein the modifications are located at the 5’ and / or 3’ nucleotides.

20. The method of claim 19, wherein the gRNA comprises the nucleotide sequence of SEQ ID NO: 14.

21. The method of any one of claims 1-20, wherein the lipid excipients comprise:(a) between 40 and 50 mol% of a cationic lipid;(b) a neutral lipid,(c) a steroid; and(d) a polymer conjugated lipid.

22. The method of claim 21, wherein the lipid excipients comprise between 45 to 48 mol%, optionally between 47.2 to 47.8 mol%, of the cationic lipid.

23. The method of claim 21 or claim 22, wherein the lipid excipients comprise between 5 and 15 mol% of the neutral lipid; optionally wherein the lipid excipients comprise between 9 and 11 mol%, optionally 10 mol%, of the neutral lipid.

24. The method of any one of claims 21-23, wherein the lipid excipients comprise between 32 and 40 mol% of the steroid or between 39 to 49 mol% of the steroid; optionally wherein the lipid excipients comprise 40 mol% of the steroid.

25. The method of any one of claims 21-24, wherein the neutral lipid is DSPC, the steroid is cholesterol, and / or the polymer conjugated lipid is a pegylated lipid.

26. The method of any one of claims 1-25, wherein the subject is a human patient diagnosed with PHI; optionally wherein the human patient is diagnosed with carrying a mutation in theAGXT gene.

27. The method of claim 26, wherein the human patient is an adult patient, optionally an adult patient 18 to 64 years of age.

28. The method of claim 26, wherein the human patient is a pediatric patient, optionally a pediatric patient 6 to 17 years of age.

29. The method of any one of claims 26-28, wherein the subject has one or more of the following features:(a) preserved kidney function at eGFR > 30 mL / min / 1.73 m2;(b) mean 24-hour urinary oxalate > 0.7 mmol / 24 hours / 1.73 m2; and (c) no evidence of systemic oxalosis.

30. The method of any one of claims 1-29, wherein the gene editing composition is administered to the subject via intravenous infusion, optionally over 2 hours.

31. The method of any one of claims 1-30, wherein the subject receives dexamethasone, antihistamine and / or acetaminophen / paracetamol prior to the administration of the gene editing composition, after the administration of the gene editing composition, or a combination thereof.

32. The method of claim 31, wherein the subject is an adult patient and receives:(a) 16-24 hours prior to the administration of the gene editing composition, dexamethasone orally or by intravenous injection; optionally at 8 or 10 mg;(b) 1-2 hours prior to the administration of the gene editing composition, (i) dexamethasone by intravenous injection, optionally at 10 mg; (ii) Hi blocker by intravenous injection, optionally diphenhydramine at 50 mg or an equivalent thereof, or Hi blocker orally, optionally cetirizine at 10 mg or an equivalent thereof; (iii) H2 blocker orally or by intravenous injection; optionally famotidine at 20 mg or an equivalent thereof; and / or (iv) acetaminophen / paracetamol by intravenous injection, optionally at 15 mg / kg (<50 kg) or 750 mg (>50 kg);(c) 6 hours after the administration of the gene editing composition, dexamethasone by intravenous injection, optionally at 4 mg; and(d) 24 hours after the administration of the gene editing composition, dexamethasone by intravenous injection, optionally at 4 mg.

33. The method of claim 31, wherein the subject is a pediatric patient and receives:(a) 16-24 hours prior to the administration of the gene editing composition, dexamethasone orally; optionally at 0.15 mg / kg up to 8 mg;(b) 1-2 hours prior to the administration of the gene editing composition, (i) dexamethasone by intravenous injection, optionally at 0.18 mg / kg up to 10 mg; (ii) Hi blocker by intravenous injection, optionally diphenhydramine at 2 mg / kg and up to 50 mg or an equivalent thereof, or Hi blocker orally, optionally cetirizine at 10 mg or an equivalent thereof; (iii) H2 blocker orally or by intravenous injection; optionally famotidine at 0.25 mg / kg and up to 20 mg or an equivalent thereof; and / or (iv)acetaminophen / paracetamol by intravenous injection, optionally at 15 mg / kg (<50 kg) or 750 mg (>50 kg);(c) 6 hours after the administration of the gene editing composition, dexamethasone by intravenous injection, optionally at 0.075 mg / kg and up to 4 mg; and (d) 24 hours after the administration of the gene editing composition, dexamethasone by intravenous injection, optionally at 0.075 mg / kg and up to 4 mg.

34. The method of any one of claims 1-33, further comprising measuring levels of GO enzyme, urinary oxalate, and / or serum glycolate after the administration of the gene editing composition.

35. The method of any one of claims 1-34, further comprising monitoring occurrence of an adverse event after the administration of the gene editing composition; and managing the adverse event when occurrence is observed.

36. A gene editing composition, which comprises:(a) a messenger RNA (mRNA) encoding a Casl2i2 polypeptide, which comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1, wherein the Casl2i2 polypeptide comprises at least amino acid substitutions D581R, I926R, and V1030G relative to SEQ ID NO: 1;(b) a guide RNA (gRNA) comprising a spacer sequence, which is specific to a target sequence of 5’-CAAAGTCTATATATGACTAT-3’ (SEQ ID NO: 10) in the HA01 gene; and(c) and lipid excipients, which comprise a cationic lipid, a zwitterionic phospholipid, cholesterol, a pegylated lipid, or a combination thereof; wherein the mRNA and gRNA are associated with the lipid excipients, and optionally wherein the lipid excipients form lipid nanoparticles (LNPs) and the mRNA and gRNA are attached to or encapsulated by the LNPs; andwherein the gene editing composition is at an amount of total RNA of about 0.4 mg / kg to about 1 mg / kg,for use in treating primary hyperoxaluria 1 (PHI) in a subject.

37. The composition for use according to claim 36, wherein the amount of total RNA does not exceed 75 mg.

38. The composition for use according to claim 36 or claim 37, wherein the amount of total RNA is about 0.4 mg / kg, about 0.7 mg / kg, or about 1.0 mg / kg.

39. The composition for use according to any of claims 36-38, wherein the Casl2i2 polypeptide comprises:(c) D581R, I926R, and V1030G relative to SEQ ID NO: 1; or(d) D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G relative to SEQ ID NO: 1.

40. The composition for use according to claim 39, wherein the Casl2i2 polypeptide comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

41. The composition for use according to claims 36-40, wherein the Casl2i2 polypeptide further comprises a first nuclear localization signal (NLS) at the N-terminus, a second nuclear localization signal (NLS) at the C-terminus, or both, of the Casl2i2 polypeptide; optionally wherein the first NLS and the second NLS are identical.

42. The composition for use according to claim 41, wherein the first NLS and / or the second NLS is set forth as KRPAATKKAGQAKKKK (SEQ ID NO: 5).

43. The composition for use according to claim 41 or claim 42, wherein the Casl2i2 polypeptide further comprises a peptide linker between the NLS and the rest of the polypeptide; optionally wherein the peptide linker is set forth as SAGGGGSGGGGSGGGGSG (SEQ ID NO: 6).

44. The composition for use according to claims 41-43, wherein the Casl2i2 polypeptide comprises the first NLS at the N-terminus and the second NLS at the C-terminus, andwherein the Casl2i2 polypeptide comprises the peptide linker connecting each of the first NLS and the second NLS to the rest of the polypeptide.

45. The composition for use according to claim 44, wherein the Casl2i2 polypeptide comprises the amino acid sequence of SEQ ID NO: 7.

46. The composition for use according to claim 45, wherein the mRNA comprises a coding sequence at least 80% identical to SEQ ID NO: 8 and encodes the amino acid sequence of SEQ ID NO: 7.

47. The composition for use according to any one of claims 36-46, wherein the mRNA comprises a 5’ cap moiety and a 3’ polyadenylation tail.

48. The composition for use according to any one of claims 36-47, wherein the mRNA comprises one or more modified nucleotides, which comprise pseudouridine; optionally wherein at least 70% of the uridine positions in the mRNA contain pseudouridines.

49. The composition for use according to claim 36, wherein the mRNA comprises the nucleotide sequence of SEQ ID NO: 23; optionally wherein the mRNA comprises a 5’ cap moiety, and pseudouridine residues at all uridine positions in the mRNA.

50. The composition for use according to any one of claims 36-49, wherein the gRNA comprises the spacer sequence set forth as 5’-CAAAGUCUAUAUAUGACUAU-3’ (SEQ ID NO: 11).

51. The composition for use according to any one of claims 36-50, wherein the gRNA further comprises a direct repeat sequence, which optionally is 23-26 nucleotide in length.

52. The composition for use according to claim 51, wherein the direct repeat sequence is 5’-AGAAAUCCGUCUUUCAUUGACGG-3’ (SEQ ID NO: 12).

53. The composition for use according to any one of claims 36-52, wherein the gRNA comprises the nucleotide sequence of 5’- AGAAAUCCGUCUUUCAUUGACGGCAAAGUCUAUAUAUGACUAU-3’ (SEQ ID NO: 13).

54. The composition for use according to any one of claims 36-53, wherein the gRNA comprises one or more modifications, which optionally comprises phosphorothioate linkage and 2’-O-methylation; optionally wherein the modifications are located at the 5’ and / or 3’ nucleotides.

55. The composition for use according to claim 54, wherein the gRNA comprises the nucleotide sequence of SEQ ID NO: 14.

56. The composition for use according to any one of claims 36-55, wherein the lipid excipients comprise:(e) between 40 and 50 mol% of a cationic lipid;(f) a neutral lipid,(g) a steroid; and(h) a polymer conjugated lipid.

57. The composition for use according to claim 56, wherein the lipid excipients comprise between 45 to 48 mol%, optionally between 47.2 to 47.8 mol%, of the cationic lipid.

58. The composition for use according to claim 56 or claim 57, wherein the lipid excipients comprise between 5 and 15 mol% of the neutral lipid; optionally wherein the lipid excipients comprise between 9 and 11 mol%, optionally 10 mol%, of the neutral lipid.

59. The composition for use according to any one of claims 56-58, wherein the lipid excipients comprise between 32 and 40 mol% of the steroid or between 39 to 49 mol% of the steroid; optionally wherein the lipid excipients comprise 40 mol% of the steroid.

60. The composition for use according to any one of claims 56-59, wherein the neutral lipid is DSPC, the steroid is cholesterol, and / or the polymer conjugated lipid is a pegylated lipid.

61. The composition for use according to any one of claims 36-60, wherein the subject is a human patient; optionally wherein the primary hyperoxaluria 1 (PHI) is caused by a mutation in theAGXT gene.

62. The composition for use according to claim 61, wherein the human patient is an adult patient, optionally an adult patient 18 to 64 years of age.

63. The composition for use according to claim 61, wherein the human patient is a pediatric patient, optionally a pediatric patient 6 to 17 years of age.

64. The composition for use according to any one of claims 61-63, wherein the subject has one or more of the following features:(a) preserved kidney function at eGFR > 30 mL / min / 1.73 m2;(b) mean 24-hour urinary oxalate > 0.7 mmol / 24 hours / 1.73 m2; and (c) no evidence of systemic oxalosis.

65. The composition for use according to claims 36-64, wherein the gene editing composition is administered to the subject via intravenous infusion, optionally over 2 hours.

66. The composition for use according to claims 36-65, wherein the subject receives dexamethasone, antihistamine and / or acetaminophen / paracetamol prior to the administration of the gene editing composition, after the administration of the gene editing composition, or a combination thereof.

67. The composition for use according to 66, wherein the subject is an adult patient and receives:(a) 16-24 hours prior to the administration of the gene editing composition, dexamethasone orally or by intravenous injection; optionally at 8 or 10 mg;(b) 1-2 hours prior to the administration of the gene editing composition, (i) dexamethasone by intravenous injection, optionally at 10 mg; (ii) Hi blocker by intravenous injection, optionally diphenhydramine at 50 mg or an equivalent thereof, or Hi blocker orally, optionally cetirizine at 10 mg or an equivalent thereof; (iii) H2 blocker orally or by intravenous injection; optionally famotidine at 20 mg or an equivalent thereof; and / or (iv) acetaminophen / paracetamol by intravenous injection, optionally at 15 mg / kg (<50 kg) or 750 mg (>50 kg);(c) 6 hours after the administration of the gene editing composition, dexamethasone by intravenous injection, optionally at 4 mg; and(d) 24 hours after the administration of the gene editing composition, dexamethasone by intravenous injection, optionally at 4 mg.

68. The composition for use according to claim 66, wherein the subject is a pediatric patient and receives:(e) 16-24 hours prior to the administration of the gene editing composition, dexamethasone orally; optionally at 0.15 mg / kg up to 8 mg;(f) 1-2 hours prior to the administration of the gene editing composition, (i) dexamethasone by intravenous injection, optionally at 0.18 mg / kg up to 10 mg; (ii) Hi blocker by intravenous injection, optionally diphenhydramine at 2 mg / kg and up to 50 mg or an equivalent thereof, or Hi blocker orally, optionally cetirizine at 10 mg or an equivalent thereof; (iii) H2 blocker orally or by intravenous injection; optionally famotidine at 0.25 mg / kg and up to 20 mg or an equivalent thereof; and / or (iv) acetaminophen / paracetamol by intravenous injection, optionally at 15 mg / kg (<50 kg) or 750 mg (>50 kg);(g) 6 hours after the administration of the gene editing composition, dexamethasone by intravenous injection, optionally at 0.075 mg / kg and up to 4 mg; and (h) 24 hours after the administration of the gene editing composition, dexamethasone by intravenous injection, optionally at 0.075 mg / kg and up to 4 mg.

69. The composition for use according to claims 36-68, further comprising measuring levels of GO enzyme, urinary oxalate, and / or serum glycolate after the administration of the gene editing composition.

70. The composition for use according to claims 36-69, further comprising monitoring occurrence of an adverse event after the administration of the gene editing composition; and managing the adverse event when occurrence is observed.

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