Compositions and methods for editing the human alpha kinase 1 (ALPK1) gene and treating rosah syndrome

Abstract

A modified adenine base editor (ABE) and a specific guide RNA (gRNA) sequence for correcting a genetic mutation in the human alpha kinase 1 (ALPK1) gene are described. The modified ABE was generated by introducing a mutation corresponding to the high fidelity (HiFi) R691A mutation previously described for Cas9 into the ABE7.10-SpRY base editor. Methods of editing a thymine mutation at nucleotide 710 of the ALPK1 gene in an isolated cell or in a subject using the disclosed ABE and gRNA are also described. In some instances, the subject has ROSAH syndrome and / or a tumor with a somatic mutation in the ALPK1 gene.

Description

[0001] 4239-112678-02

[0002] COMPOSITIONS AND METHODS FOR EDITING THE HUMAN ALPHA KINASE

[0003] 1 (ALPK1) GENE AND TREATING ROSAH SYNDROME

[0004] CROSS REFERENCE TO RELATED APPLICATIONS

[0005] This application claims the benefit of U.S. Application No. 63 / 733,836, filed December 13, 2024, which is herein incorporated by reference in its entirety.

[0006] FIELD

[0007] This disclosure concerns guide RNA (gRNA) targeting the human alpha kinase 1 (ALPK1) gene and a modified adenine base editor for use in methods of editing a mutation in the ALPK1 gene (c.710C>T, p.Thr237Met) and in methods of treating subjects harboring the ALPK1 mutation.

[0008] ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0009] This invention was made with government support under Z1 AAI000644 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0010] INCORPORATION OF ELECTRONIC SEQUENCE LISTING

[0011] The electronic sequence listing, submitted herewith as an XML file named 4239- 112678-02. xml (25,846 bytes), created on December 4, 2025, is herein incorporated by reference in its entirety.

[0012] BACKGROUND

[0013] A missense mutation (c.710C>T) in the human alpha kinase 1 (ALPK1) gene is associated with an autosomal dominant ocular systemic disorder known as ROSAH syndrome. This syndrome presents with clinical features that include retinal dystrophy, optic nerve edema, splenomegaly, anhidrosis, and migraine headache. The majority of patients with ROSAH syndrome exhibit at least one inflammatory feature, such as recurrent fever, malaise, episodic abdominal pain, headaches, transient cytopenia and uveitis with retinal vasculitis. Other clinical manifestations include optic disc elevation, blindness, splenomegaly, gastrointestinal symptoms, arthralgia, arthritis, dental caries, dry mouth, anhidrosis / hypohidrosis, and the inability to lactate. A need exists for therapeutic interventions for patients suffering from ROSAH syndrome. 4239-112678-02

[0014] SUMMARY

[0015] Described herein are compositions and methods for correcting a genetic mutation in the human alpha kinase 1 (ALPK1) gene (C.710OT, p.Thr237Met) in a cell or subject. In particular, disclosed is a specific gRNA and base editor combination that leads to highly efficient correction of the C710T mutation in the ALPK1 gene with little to no off-target effects.

[0016] Provided is a modified adenine base editor (ABE) generated by incorporating the CRISPR / Cas9 high-fidelity (“HiFi”) R691 A mutation into the ABE7.10-SpRY base editor. The HiFi mutation prevents off-target effects of ALPLK1 gene editing. In some aspects, the amino acid sequence of the modified ABE includes or consists of SEQ ID NO: 14. Nucleic acid molecules and vectors (such as viral vectors) encoding the modified ABE are also described.

[0017] Also provided is a gRNA targeting the ALPK1 gene. In some aspects, the nucleotide sequence of the gRNA includes SEQ ID NO: 1.

[0018] Further provided are methods of editing a thymine mutation at nucleotide 710 of the human ALPK1 gene in an isolated cell. In some aspects, the method includes administering to the isolated cell (i) a gRNA that includes the nucleotide sequence of SEQ ID NO: 1, or a nucleic acid molecule encoding a gRNA that includes the nucleotide sequence of SEQ ID NO: 1; and

[0019] (ii) a modified base editor that includes the amino acid sequence of SEQ ID NO: 14, or a nucleic acid molecule encoding the modified base editor. In some examples, the isolated cell is an induced pluripotent stem cell (iPSC) or a hematopoietic stem cell.

[0020] Also provided is a method of correcting a genetic mutation in a subject, wherein the genetic mutation is a cytosine to thymine mutation at nucleotide 710 of the human ALPK1 gene. In some aspects, the method includes administering to the subject (i) a gRNA that includes the nucleotide sequence of SEQ ID NO: 1, or a nucleic acid molecule encoding a gRNA that includes the nucleotide sequence of SEQ ID NO: 1; and (ii) a modified base editor having the amino acid sequence of SEQ ID NO: 14, or a nucleic acid molecule encoding the modified base editor. In some examples, the subject has ROSAH syndrome. In other examples, the subject has a tumor with a somatic mutation in the ALPK1 gene. In some aspects of the method, administration of the gRNA and modified base editor (or their encoding nucleic acid molecules) includes intravenous administration, intraocular administration, portal vein administration, intratumoral administration, or retroductal cannulation of a salivary gland. 4239-112678-02

[0021] Further provided are ex vivo methods of editing a thymine mutation at nucleotide 710 of the ALPK1 gene in iPSCs. In some aspects, the method includes administering to the iPSCs a gRNA that includes the nucleotide sequence of SEQ ID NO: 1, or a nucleic acid molecule encoding a gRNA of SEQ ID NO: 1 ; and a modified base editor having the amino acid sequence of SEQ ID NO: 14, or a nucleic acid molecule encoding the modified base editor; differentiating the iPSCs to differentiated cells; and administering the differentiated cells to a subject harboring a cytosine to thymine mutation at nucleotide 710 of the human ALPK1 gene. In some examples, the method further includes isolating cells from the subject harboring a cytosine to thymine mutation at nucleotide 710 of the human ALPK1 gene and reprogramming the cells into iPSCs.

[0022] The foregoing and other features of this disclosure will become more apparent from the following detailed description of several aspects which proceeds with reference to the accompanying figures.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIGS. 1A-1C: Comparison between ALPK1 -targeted A5 and A4 guide RNAs (gRNAs) using the adenine base editor ABE7.10-SpRY to correct the ALPK1 C.710OT mutation (p.Thr237Met) in induced pluripotent stem cells (iPSCs) derived from a ROSAH patient. (FIG. 1A) Sanger sequencing of the ALPK1 c.710C>T mutation in the patient iPSCs, demonstrating the presence of the heterozygous C>T mutation at the c.710 position. (FIG. IB) Sequence following editing of patient iPSCs using ABE7.10-SpRY with the A5 guide RNA (SEQ ID NO: 1). (FIG. 1C) Sequence following editing of patient iPSCs using ABE7. 10-SpRY with the A4 guide RNA (SEQ ID NO: 2). The A5 guide RNA produced the highest levels of correction without bystander edits in the ALPK1 gene.

[0025] FIGS. 2A-2D: Comparison of different ABE editors using the ALPK1 A5 guide RNA to correct the ALPK1 C.710OT mutation (p.Thr237Met) in patient iPSCs. (FIG. 2A) Sanger sequencing of the ALPK1 C.710OT mutation in patient iPSCs (without editing). (FIG. 2B) Sequence following editing of patient iPSCs with AB E7.10-SpRY and the A5 guide RNA. (FIG. 2C) Sequence following editing of patient iPSCs with ABE8e-HiFi-SpRY and the A5 guide RNA. (FIG. 2D) Sequence following editing of patient iPSCs with ABE9- SpRY and the A5 guide RNA. Use of the ABE7. 10-SpRY base editor produced the highest levels of correction without bystander edits in the ALPK1 gene. 4239-112678-02

[0026] FIGS. 3A-3B: Whole genome sequencing data of edited versus unedited iPSCs from a ROSAH patient. Whole genome sequencing of two biological replicates of patient iPSCs edited with ABE7. 10-SpRY and the A5 guide RNA revealed a number of off-target edits occurring either in inter-gene regions (IGRs), intronic regions within genes, downstream untranslated regions (3' UTRs) or upstream flanking regions of genes (5' Flank), as well as a missense mutation within the FGFR2 gene that occurred with a high frequency in both edited iPSC samples, and a neighboring silent mutation in the same region of the FGFR2 gene. These FGFR2 mutations occurred within a region with 17 out of 20 matching nucleotides to the ALPK1 sequence targeted by the A5 guide RNA, confirming that these were off-target edits caused by the combination of AB E7.10-SpRY and the A5 guide RNA.

[0027] FIGS. 4A-4D: Comparison of different ABEs using the ALPK1 A5 gRNA for inducing FGFR2 off-target edits. (FIG. 4A) Unedited patient iPSCs lack the FGFR2 off- target mutation. (FIG. 4B) Patient iPSCs edited with ABE7.10-SpRY and the A5 guide RNA show both the FGFR2 off-target silent (c.1683) and missense (c.1681) mutations. (FIG. 4C) Patient iPSCs edited with ABE8e-HiFi-SpRY and the A5 guide RNA show even higher levels of the FGFR2 off-target silent and missense FGFR2 mutations. (FIG. 4D) Patient iPSCs edited with ABE9-SpRY and the A5 guide RNA have a lower level of the FGFR2 off- target missense mutation and no off-target silent mutations.

[0028] FIG. 5: Plasmid map of ABE7. 10-HiFi-SpRY. A R691A mutation in Cas9 confers high fidelity (reduced off-target activity). The R691A HiFi mutation was introduced into the nCas9-SpRY portion of the existing ABE7.10 base editor expression plasmid (Addgene plasmid # 140003), and AB E7.10-HiFi-SpRY mRNA was synthesized for analysis.

[0029] FIGS. 6A-6F: Comparison between ABE7. 10-SpRY and AB E7.10-HiFi-SpRY for inducing FGFR2 off-target edits using the AEPK1 A5 gRNA. (FIGS. 6A-6B) Sanger sequencing of the FGFR2 off-target site in unedited iPSCs from two different ROSAH patients that share the ALPK1 C.710OT mutation. (FIG. 6C) Sanger sequencing of iPSCs from one patient edited with the regular-fidelity ABE7.10-SpRY base editor and A5 guide RNA. (FIGS. 6D-6F) Three replicates of patient iPSCs edited with the high-fidelity ABE7. 10-HiFi-SpRY base editor and A5 guide RNA.

[0030] FIGS. 7A-7F: Comparison between ABE7.10-SpRY and ABE7.10-HiFi-SpRY for correcting the ALPK1 C.710OT mutation (p.Thr237Met) in patient iPSCs using the ALPK1 A5 gRNA. (FIGS. 7A-7B) Sanger sequencing confirming the presence of the ALPK1 c.710C>T mutation in iPSCs from two different ROSAH patients. (FIG. 7C) Sanger sequencing data of patient iPSCs edited with the regular-fidelity ABE7.10-SpRY base editor 4239-112678-02 and A5 guide RNA. (FIGS. 7D-7F) Three replicates of patient iPSCs edited with the high- fidelity ABE7.10-HiFi-SpRY base editor and A5 guide RNA.

[0031] FIG. 8: Table summarizing whole genome sequencing analysis following editing using the ALPK1 A5 gRNA with either ABE7.10-SpRY or ABE7.10-HiFi-SpRY. Whole genome sequencing incorporates a greater number of DNA sequencing reads (~40 reads per cell sample) than Sanger sequencing (1 read per cell sample), providing a more sensitive and in-depth assessment of correction efficiency. Based on the whole genome sequencing data, the level of correction of the ALPK1 mutation in the three replicate iPSC correction samples was 68-94% (compared to the -68-76% correction detected for these three samples by Sanger sequencing in FIGS. 7D-7F).

[0032] SEQUENCES

[0033] The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and single letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:

[0034] SEQ ID NO: 1 is the nucleotide sequence of the target-specific portion of the ALPK1 A5 gRNA.

[0035] CGACAUGGAGAGGCCCUGUA

[0036] SEQ ID NO: 2 is the nucleotide sequence of the target-specific portion of the ALPK1 A4 gRNA.

[0037] GACAUGGAGAGGCCCUGUAA

[0038] SEQ ID NO: 3 is an unedited genomic sequence from a ROSAH-1 patient (FIG. 1). GTGTTTTTCTTACAGGGCCTCTCCAYGTCGCTAGGTATACTGGCAG

[0039] SEQ ID NO: 4 is an edited genomic sequence from a ROSAH-1 patient (FIG. 1). GTGTTTTTCTTACAGGGCCTCTCCACGTCGCTAGGTATACTGGCAG

[0040] SEQ ID NO: 5 is an edited genomic sequence from a ROSAH-1 patient (FIG. 1). GTGTTTTTCTTACAGGGCCTCYCCAYGTCGCTAGGTATACTGGCAG

[0041] SEQ ID NO: 6 is an edited genomic sequence from a ROSAH-1 patient (FIG. 2). GTGTTTTTCTTACAGGGCCTCYCCACGTCGCTAGGTATACTGGCAG

[0042] SEQ ID NO: 7 i s the nucleotide sequence of a portion of the A5 gRNA (FIG. 3B). GACATGGAGAGGCCCTGT

[0043] SEQ ID NO: 8 is human genomic DNA sequence (FIG. 3B). 4239-112678-02

[0044] GACATAGAGAGGCCCTGT

[0045] SEQ ID NO: 9 is an unedited genomic sequence from a ROS AH- 1 patient (FIGS. 4 and 6).

[0046] CTTCCTCAACAGGGCCTCTCTATGTCATAGTTGAGTATGCCTC

[0047] SEQ ID NO: 10 is an edited genomic sequence from a ROSAH-1 patient (FIGS. 4 and 6).

[0048] CTTCCTCAACAGGGCCTCTCYATGTCATAGTTGAGTATGCCTC

[0049] SEQ ID NO: 11 is an edited genomic sequence from a ROSAH-1 patient (FIG. 4).

[0050] CTTCCTCAACAGGGCCTCTCYAYGTCATAGTTGAGTATGCCTC

[0051] SEQ ID NO: 12 is the genomic DNA sequence that binds the ALPK1 A5 gRNA.

[0052] TACAGGGCCTCTCCATGTCG

[0053] SEQ ID NO: 13 is the amino acid sequence of human wild-type ALPK1 (deposited under GENBANK Accession No. NP_001095876.1). Threonine 237 is underlined.

[0054] MNNQKVVAVLLQECKQVLDQLLLEAPDVSEEDKSEDQRCRALLPSELRTLIQEAKE

[0055] MKWPFVPEKWQYKQAVGPEDKTNLKDVIGAGLQQLLASLRASILARDCAAAAAIVF

[0056] LVDRFLYGLDVSGKLLQVAKGLHKLQPATPIAPQVVIRQARISVNSGKLLKAEYILSS

[0057] LISNNGATGTWLYRNESDKVLVQSVC1QIRGQILQKLGMWYEAAEL1WAS1VGYLAL

[0058] PQPDKKGLSTSLGILADIFVSMSKNDYEKFKNNPQINLSLLKEFDHHLLSAAEACKLA

[0059] AAFSAYTPLFVLTAVNIRGTCLLSYSSSNDCPPELKNLHLCEAKEAFEIGLLTKRDDEP

[0060] VTGKQELHSFVKAAFGLTTVHRRLHGETGTVHAASQLCKEAMGKLYNFSTSSRSQD

[0061] REALSQEVMSVIAQVKEHLQVQSFSNVDDRSYVPESFECRLDKLILHGQGDFQKILD

[0062] TYSQHHTSVCEVFESDCGNNKNEQKDAKTGVCITALKTEIKNIDTVSTTQEKPHCQR

[0063] DTGISSSLMGKNVQRELRRGGRRNWTHSDAFRVSLDQDVETETEPSDYSNGEGAVF

[0064] NKSLSGSQTSSAWSNLSGFSSSASWEEVNYHVDDRSARKEPGKEHLVDTQCSTALSE

[0065] ELENDREGRAMHSLHSQLHDLSLQEPNNDNLEPSQNQPQQQMPLTPFSPHNTPGIFL

[0066] APGAGLLEGAPEGIQEVRNMGPRNTSAHSRPSYRSASWSSDSGRPKNMGTHPSVQK

[0067] EEAFEIIVEFPETNCDVKDRQGKEQGEEISERGAGPTFKASPSWVDPEGETAESTEDA

[0068] PLDFHRVLHNSLGNISMLPCSSFTPNWPVQNPDSRKSGGPVAEQGIDPDASTVDEEG

[0069] QLLDSMDVPCTNGHGSHRLCILRQPPGQRAETPNSSVSGNILFPVLSEDCTTTEEGNQ

[0070] PGNMLNCSQNSSSSSVWWLKSPAFSSGSSEGDSPWSYLNSSGSSWVSLPGKMRKEIL

[0071] EARTLQPDDFEKLLAGVRHDWLFQRLENTGVFKPSQLHRAHSALLLKYSKKSELWT

[0072] AQETIVYLGDYLTVKKKGRQRNAFWVHHLHQEEILGRYVGKDYKEQKGLWHHFTD

[0073] VERQMTAQHYVTEFNKRLYEQNIPTQIFYIPSTILLILEDKTIKGCISVEPYILGEFVKLS

[0074] NNTKVVKTEYKATEYGLAYGHFSYEFSNHRDVVVDLQGWVTGNGKGLIYLTDPQI

[0075] HSVDQKVFTTNFGKRGIFYFFNNQHVECNEICHRLSLTRPSMEKPCT

[0076] SEQ ID NO: 14 is the amino acid sequence of the modified base editor

[0077] ABEmax(7.10)-HiFi-SpRY. The R691A HiFi substitution is underlined.

[0078] MKRTADGSEFESPKKKRKVSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHN

[0079] NRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGA

[0080] MIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR

[0081] MRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEY

[0082] WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQ 4239-112678-02

[0083] GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV LHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSG SETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGN TDRHSIKKNLIGALLFDSGETAERTRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDD

[0084] SFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL

[0085] IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAI

[0086] LSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKD

[0087] TYDDDEDNELAQIGDQYADLFEAAKNLSDA1EESDIERVNTE1TKAPLSASM1KRYDE

[0088] HHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD

[0089] GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKI

[0090] LTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK

[0091] NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTN

[0092] RKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENED

[0093] ILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD

[0094] KQSGKTILDFLKSDGFANANFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG

[0095] SPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEE

[0096] GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVP

[0097] QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN

[0098] LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVIT

[0099] LKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY

[0100] KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI

[0101] VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIRPKRNSDKLIARKKDWDP KKYGGFLWPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK GYKEVKKDLIIKLPKYSLFELENGRKRMLASAKQLQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKH YLDEIIEQISEFS KRVILAD ANLDKVLS AYNKHRD

[0102] KPIREQAENIIHLFTLTRLGAPRAFKYFDTTIDPKQYRSTKEVLDATLIHQSITGLYETR IDLSQLGGD

[0103] SEQ ID NO: 15 is a nucleic acid sequence encoding ABEmax(7.10)-HiFi-SpRY.

[0104] ATGAAACGGACAGCCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAGCGGAA AGTCTCTGAAGTCGAGTTTAGCCACGAGTATTGGATGAGGCACGCACTGACCCT GGCAAAGCGAGCATGGGATGAAAGAGAAGTCCCCGTGGGCGCCGTGCTGGTGC ACAACAATAGAGTGATCGGAGAGGGATGGAACAGGCCAATCGGCCGCCACGAC

[0105] CCTACCGCACACGCAGAGATCATGGCACTGAGGCAGGGAGGCCTGGTCATGCAG AATTACCGCCTGATCGATGCCACCCTGTATGTGACACTGGAGCCATGCGTGATGT GCGCAGGAGCAATGATCCACAGCAGGATCGGAAGAGTGGTGTTCGGAGCACGG GACGCCAAGACCGGCGCAGCAGGCTCCCTGATGGATGTGCTGCACCACCCCGGC

[0106] ATGAACCACCGGGTGGAGATCACAGAGGGAATCCTGGCAGACGAGTGCGCCGC

[0107] CCTGCTGAGCGATTTCTTTAGAATGCGGAGACAGGAGATCAAGGCCCAGAAGAA GGCACAGAGCTCCACCGACTCTGGAGGATCTAGCGGAGGATCCTCTGGAAGCGA GACACCAGGCACAAGCGAGTCCGCCACACCAGAGAGCTCCGGCGGCTCCTCCGG AGGATCCTCTGAGGTGGAGTTTTCCCACGAGTACTGGATGAGACATGCCCTGAC

[0108] CCTGGCCAAGAGGGCACGCGATGAGAGGGAGGTGCCTGTGGGAGCCGTGCTGG TGCTGAACAATAGAGTGATCGGCGAGGGCTGGAACAGAGCCATCGGCCTGCACG ACCCAACAGCCCATGCCGAAATTATGGCCCTGAGACAGGGCGGCCTGGTCATGC

[0109] AGAACTACAGACTGATTGACGCCACCCTGTACGTGACATTCGAGCCTTGCGTGA TGTGCGCCGGCGCCATGATCCACTCTAGGATCGGCCGCGTGGTGTTTGGCGTGA GGAACGCAAAAACCGGCGCCGCAGGCTCCCTGATGGACGTGCTGCACTACCCCG

[0110] GCATGAATCACCGCGTCGAAATTACCGAGGGAATCCTGGCAGATGAATGTGCCG CCCTGCTGTGCTATTTCTTTCGGATGCCTAGACAGGTGTTCAATGCTCAGAAGAA 4239-112678-02

[0111] GGCCCAGAGCTCCACCGACTCCGGAGGATCTAGCGGAGGCTCCTCTGGCTCTGA

[0112] GACACCTGGCACAAGCGAGAGCGCAACACCTGAAAGCAGCGGGGGCAGCAGCG

[0113] GGGGGTCAGACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACTCTGTGG

[0114] GCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGC

[0115] TGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGT

[0116] TCGACAGCGGCGAAACAGCCGAGAGAACCCGGCTGAAGAGAACCGCCAGAAGA

[0117] AGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAAC

[0118] GAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTG

[0119] GTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGA

[0120] CGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACT

[0121] GGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCA

[0122] CATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAA

[0123] CAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTT

[0124] CGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGC

[0125] CAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCG

[0126] AGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCC

[0127] CCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCA

[0128] AGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGT

[0129] ACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCG

[0130] ACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGA

[0131] TCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGC

[0132] GGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACG

[0133] GCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCA

[0134] TCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGA

[0135] ACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCC

[0136] ACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTT

[0137] ACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCA

[0138] TCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGA

[0139] CCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGAC

[0140] AAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAAC

[0141] CTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACC

[0142] GTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCC

[0143] CGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGAC

[0144] CAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCG

[0145] AGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCT

[0146] GGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAA

[0147] TGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGA

[0148] GGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGA

[0149] CAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGA

[0150] GCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTG

[0151] GATTTCCTGAAGTCCGACGGCTTCGCCAACGCGAACTTCATGCAGCTGATCCACG

[0152] ACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGG

[0153] GCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGA

[0154] AGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGC

[0155] CGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCAC

[0156] CCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGC

[0157] ATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCA

[0158] GCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTA

[0159] CGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATAT

[0160] CGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAG 4239-112678-02

[0161] AAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGA

[0162] AGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGA

[0163] GAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGAT

[0164] AAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCA

[0165] CGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAA

[0166] GCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTT

[0167] CCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGC

[0168] CCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCC

[0169] TAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAA

[0170] GATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTT

[0171] CTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGA

[0172] GATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGT

[0173] GGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAG

[0174] TGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCT

[0175] ATCAGACCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGA

[0176] CCCTAAGAAGTACGGCGGCTTCCTGTGGCCCACCGTGGCCTATTCTGTGCTGGTG

[0177] GTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCT

[0178] GCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTT

[0179] TCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGC

[0180] CTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTG

[0181] CCAAGCAGCTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACT

[0182] TCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATG

[0183] AGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCG

[0184] AGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACA

[0185] AAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCG

[0186] AGAATATCATCCACCTGTTTACCCTGACCAGACTGGGAGCCCCTAGAGCCTTCAA

[0187] GTACTTTGACACCACCATCGACCCCAAGCAGTACAGAAGCACCAAAGAGGTGCT

[0188] GGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGA

[0189] CCTGTCTCAGCTGGGAGGTGAC

[0190] DETAILED DESCRIPTION

[0191] I. Abbreviations

[0192] ABE adenine base editor

[0193] ALPK1 alpha kinase 1

[0194] DSB double-strand DNA break

[0195] FGFR2 fibroblast growth factor receptor 2 gRNA guide RNA

[0196] HDR homology-directed repair

[0197] HiFi high fidelity iPSC induced pluripotent stem cell

[0198] PAM protospacer-adjacent motif

[0199] ROSAH retinal dystrophy, optic nerve edema, splenomegaly, anhidrosis and migraine headache 4239-112678-02

[0200] IL Summary of Terms

[0201] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “a mutation” includes singular or plural mutations and can be considered equivalent to the phrase “at least one mutation.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:

[0202] Adenine base editor (ABE): A protein that mediates conversion of A T base pairs to G- C base pairs in genomic DNA. Directed evolution and protein engineering have produced 7thgeneration ABEs (such as ABE7.10) that are able to efficiently convert target A T to G C base pairs, with high product purity (>99.9%), very low rates of indels (<0. 1%), and without creating double-stranded DNA breaks (DSBs) (Gaudelli et al., Nature 551(7681):464-471, 2017).

[0203] Administration: To provide or give a subject an agent, such as a therapeutic agent (e.g. a gRNA and / or a base editor), by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, intraductal, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes. In some aspects herein, administration is by intravenous administration, intraocular administration, portal vein administration, intratumoral administration, or retroductal cannulation of a salivary gland.

[0204] Alpha kinase 1 (ALPK1): A member of the alpha kinase family, which is a class of atypical protein kinases with a unique catalytic domain architecture (Middelbeek et al., Cell Molec Life Sci 67:875-890, 2010). The ALPK1 gene is located on human chromosome 4q25 at genomic coordinates (GRch38): 4:112,297,369-112,442,621. 4239-112678-02

[0205] Base editor: A protein that is capable of making a modification to a nucleotide base (e.g., A, T, C, G, or U) within a nucleic acid sequence (e.g.. DNA or RNA). Base editors include a deaminase enzyme that introduces a single nucleotide polymorphism by chemically altering the target DNA sequence without generating a DSB. Deamination involves the removal of an amino group from the nucleotide, which after DNA repair or replication results in the installation of a new base. DNA base editors include a catalytically disabled nuclease fused to a nucleobase deaminase enzyme and, in some cases, a DNA glycosylase inhibitor. RNA base editors achieve analogous changes using components that target RNA. Base editors directly convert one base or base pair into another, enabling the efficient installation of point mutations in non-dividing cells without generating excess undesired editing byproducts. Cytosine base editors (CBEs) substitute a cytosine base for a thymine. Adenine base editors (ABEs) substitute adenine with guanine. For a review, see Rees and Liu, Nat Rev Genet 19(12):770-788, 2018.

[0206] Biodegradable scaffold: A temporary, porous framework used in regenerative medicine to support new tissue growth, proliferation and / or differentiation. Biodegradable scaffolds are made of materials (biodegradable polymers) that break down naturally in the body. In some examples, the biodegradable scaffold is comprised of poly(lactic-co-glycolic acid) (PLGA) (see Gupta et al., JCI Insight 10(10):el79246, 2025).

[0207] Codon-optimized: A nucleic acid molecule encoding a protein can be codon- optimized for expression of the protein in a particular organism by including the codon most likely to encode a particular amino acid at each position of the sequence. Codon usage bias is the difference in the frequency of occurrence of synonymous codons (encoding the same amino acid) in coding DNA. A codon is a series of three nucleotides (a triplet) that encodes a specific amino acid residue in a polypeptide chain or for the termination of translation. There are 20 different naturally -occurring amino acids, but 64 different codons (61 codons encoding for amino acids plus 3 stop codons). Thus, there is degeneracy because one amino acid can be encoded by more than one codon. A nucleic acid sequence can be optimized for expression in a particular organism (such as a human) by evaluating the codon usage bias in that organism and selecting the codon most likely to encode a particular amino acid. Multivariate statistical methods, such as correspondence analysis and principal component analysis, are widely used to analyze variations in codon usage. Computer programs are available to implement the statistical analyses related to codon usage, such as Codon W, GCUA, and INCA. 4239-112678-02

[0208] Guide RNA (gRNA): A polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a base editor to the target sequence. gRNAs include two components: a target-specific CRISPR RNA (crRNA) sequence and an auxiliary trans-acting RNA (tracrRNA) sequence. The tracrRNA sequence serves as a binding scaffold for Cas9 and is not target-specific. In some aspects, the degree of complementarity between the crRNA portion of the gRNA sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman- Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some aspects, the crRNA portion of a gRNA is about, or at least about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length, such as 15-25 nucleotides (e.g., 18-22 nucleotides). In particular examples, the crRNA sequence of the gRNA is 20 nucleotides in length. In some aspects, a gRNA sequence (including both crRNA and tracrRNA) is about, or at least about, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 or 120 nucleotides in length, such as about 90-110, about 95-105, or about 97-103 nucleotides in length. In particular examples, the gRNA sequence is 100 nucleotides in length. An exemplary gRNA sequence includes the target-specific nucleic acid sequence set forth herein as SEQ ID NO: 1 (A5 gRNA). In some instances, the gRNA includes one or more modified bases or chemical modifications (e.g., see Latorre et al., Angewandte Chemie 55:3548-50, 2016). In specific examples, the A5 gRNA includes the following modifications: mC*mG*mA*CAUGGAGAGGCCCUGUA (SEQ ID NO: 1), wherein m = 2’-O-methyl and * = 3’ phosphorothioate intemucleotide linkage.

[0209] Hematopoietic stem cell: A multipotent cell capable of developing into all types of blood cells, including white blood cells (e.g., lymphocytes, monocytes, neutrophils, basophils, eosinophils), red blood cells and platelets. Hematopoietic stem cells are found in the peripheral blood and the bone marrow.

[0210] Induced pluripotent stem cell (iPSC): A cell derived from a somatic cell (such as a skin or blood cell) that has been reprogrammed back to an embryonic-like pluripotent state (see, e.g., Cerneckis etal., Signal Transduct Target Ther 9( 1): 112, 2024). 4239-112678-02

[0211] Isolated: An “isolated” biological component (such as a nucleic acid or a cell) has been substantially separated, produced apart from, or purified away from other biological components in the cell or tissue of an organism in which the component occurs, such as other cells, chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and proteins.

[0212] Lipid nanoparticle: Small spherical particles composed of lipids that are suitable for delivery of therapeutic agents, such as nucleic acid molecules (e.g., mRNA) and other pharmaceutical agents. Exemplary lipid nanoparticles are well-known to the skilled person (see, e.g., Hou et al., Nat Rev Mater 6(12):1078-1094, 2021).

[0213] Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0214] Pharmaceutically acceptable carrier: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington: The Science and Practice of Pharmacy, 23rded., London, UK: Academic Press, 2020, describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds, molecules or agents. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0215] Promoter: An array of nucleic acid control sequences which direct transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of 4239-112678-02 transcription. A promoter also optionally includes distal enhancer or repressor elements. A “constitutive promoter’’ is a promoter that is continuously active and is not subject to regulation by external signals or molecules. In contrast, the activity of an “inducible promoter” is regulated by an external signal or molecule (for example, a transcription factor).

[0216] Retinal dystrophy, optic nerve edema, splenomegaly, anhidrosis and migraine headache (ROSAH) syndrome: A syndrome caused by mutations in the human alpha kinase 1 (ALPK1) gene, such as (c.710C>T). ROSAH syndrome presents with clinical features that include retinal dystrophy, optic nerve edema, splenomegaly, anhidrosis, and migraine headache (Williams et al., Genet Med 21 (9):2103-2115, 2019). The majority of patients with ROSAH syndrome exhibit at least one inflammatory feature, such as recurrent fever, malaise, episodic abdominal pain, headaches, transient cytopenia and uveitis with retinal vasculitis. Other clinical manifestations include optic disc elevation, blindness, splenomegaly, gastrointestinal symptoms, arthralgia, arthritis, dental caries, dry mouth, anhidrosis / hypohidrosis, and the inability to lactate (Kozycki et al., Ann Rheum Dis 81:1453- 1464, 2022).

[0217] Retinal pigmented epithelium (RPE): Pigmented cells that support the photoreceptors in the retina. RPE forms a barrier between the retina and the underlying choroid.

[0218] Ribonucleoprotein (RNP) complex: A molecular structure made up of RNA and protein.

[0219] Sequence identity: The identity or similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are.

[0220] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations. 4239-112678-02

[0221] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.

[0222] Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals. In some aspects herein, the subject has a mutation in the ALPK1 gene. In some examples, the subject has ROSAH syndrome.

[0223] Vector: An entity containing a nucleic acid molecule (such as a DNA or RNA molecule) bearing a promoter(s) that is operationally linked to the coding sequence of a protein of interest and can express the coding sequence. Non-limiting examples include a naked or packaged (lipid and / or protein) DNA, a naked or packaged RNA, a subcomponent of a vims or bacterium or other microorganism that may be replication-incompetent, or a vims or bacterium or other microorganism that may be replication-competent. A vector is sometimes referred to as a constmct. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art. Viral vectors are recombinant nucleic acid vectors having at least some nucleic acid sequences derived from one or more vimses. In some aspects, the viral vector is a lentivims vector, an adeno-associated vims (AAV) vector or an adenovirus vector.

[0224] III. Introduction

[0225] The present disclosure describes the successful and efficient gene editing of induced pluripotent stem cells (iPSCs) generated from cells of individuals with a mutation in the ALPK1 gene (c.710C>T, [p.Thr237Met]), which is associated with ROSAH syndrome. The specific combination of a gRNA sequence targeting ALPK1 (the A5 gRNA; SEQ ID NO: 1) and a modified base editor (ABEmax(7.10)-HiFi-SpRY; SEQ ID NO: 14) led to highly efficient correction with no bystander or off-target edits. The A5 guide RNA contains the targeting sequence CGACAUGGAGAGGCCCUGUA (SEQ ID NO: 1), which targets the A nucleotide in position 5 for base editing of A to G to correct the ALPK1 p.Thr237Met mutation. The ABEmax(7.10)-HiFi-SpRY base editor was developed by incorporation of the CRISPR-Cas9 HiFi R691A mutation (Vakulskas et al., Nat Med 24(8): 1216-1224, 2018) 4239-112678-02 conferring high-fidelity (HiFi) editing activity to the previously developed ABEmax(7.10)- SpRY base editor (Addgene plasmid #140003) to reduce off-target edits.

[0226] Current therapeutic approaches for inherited diseases of retinal degeneration have largely focused on gene replacement involving loss of function, but such approaches are not thought to be effective in diseases of over-activation such as those caused by the ALPK1 p.Thr237Met mutation. More recently, gene correction with CRISPR-Cas9 has been pursued in some ophthalmologic diseases but this process induces the formation of double-strand DNA breaks (DSBs) at the target site, which presents the risk of undesired indels or other genomic edits. The need for homology-directed repair (HDR) limits the applicability of CRISPR-Cas9 methods in non-dividing cells, such as retinal pigmented epithelial cells. In contrast, the methods disclosed herein utilize a targeted base editor to correct the pathogenic mutation without the generation of DSBs or the need for HDR. The disclosed methods can be used in patients with diseases associated with the heterozygous C710T mutation in human ALPK1, such as retinal degeneration, dental decay in the setting of hyposalivation, tumors harboring the ALPK1 mutation, or any disease state associated with ROSAH syndrome.

[0227] IV. Modified Base Editor and Guide RNA for Correcting ALPK1 Mutation

[0228] The present disclosure provides compositions for correcting a genetic mutation in the human alpha kinase 1 (ALPK1) gene (c.71QOT, p.Thr237Met), a heterozygous mutation found in patients with ROSAH syndrome. In particular, disclosed is a gRNA sequence and a modified adenine base editor (ABE) combination that provide highly efficient correction of the C710T mutation in the ALPK1 gene with little to no off-target / bystander effects.

[0229] Provided herein is a modified, high-fidelity (HiFi) ABE that exhibits efficient gene editing without inducing off-target edits. In some aspects, the ABE incorporates the R691A HiFi mutation previously described for Cas9 (Vakulskas et al., Nat Med 24(8): 1216-1224, 2018). In some examples, the amino acid sequence of the modified ABE includes or consists of SEQ ID NO: 14 with no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid substitution, such as a conservative amino acid substitution, while retaining the HiFi mutation (in SEQ ID NO: 14, the HiFi mutation is located at residue 1105). In specific examples, the amino acid sequence of the modified ABE includes or consists of SEQ ID NO: 14.

[0230] Also provided herein are nucleic acid molecules encoding the modified ABE. In some aspects, the nucleic acid molecule is codon-optimized for expression in human cells. In 4239-112678-02 some aspects, the nucleic acid molecule encoding the modified ABE is operably linked to a promoter, such as a cytomegalovirus (CMV) promoter. In some aspects, the nucleotide sequence encoding the modified ABE is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 15. In some examples, the nucleotide sequence encoding the modified ABE includes or consists of SEQ ID NO: 15.

[0231] Further provided are vectors that include a nucleic acid molecule disclosed herein. In some aspects, the vector is a plasmid vector. In other aspects, the vector is a viral vector. In some examples, the viral vector is an adenovirus vector, an adeno-associated virus (AAV) vector, or a lentivirus vector. In some aspects, the vector further includes a nucleic acid sequence encoding a guide RNA (gRNA). In some examples, the nucleotide sequence of the gRNA includes SEQ ID NO: 1.

[0232] Also provided herein are guide RNAs (gRNAs) targeting the human ALPK1 gene. In some aspects, the nucleotide sequence of the gRNA includes SEQ ID NO: 1 with no more than 4, no more than 3, no more than 2, or no more than 1 nucleotide substitution(s). In some examples, the nucleotide sequence of the gRNA includes SEQ ID NO: 1 and up to 100, up to 90, up to 80, up to 70, up to 60, up to 50, up to 40, up to 30, up to 20, or up to 10 additional nucleotide(s) at the 5' end of SEQ ID NO: 1 and / or up to 100, up to 90, up to 80, up to 70, up to 60, up to 50, up to 40, up to 30, up to 20, or up to 10 additional nucleotide(s) at the 3' end of SEQ ID NO: 1. In specific examples, the total length of the gRNA is about 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 or 120 nucleotides in length, such as about 90-110, about 95-105, or about 97-103 nucleotides in length. In one non-limiting example, the gRNA sequence is 100 nucleotides in length.

[0233] Further provided are vectors that encode a gRNA disclosed herein. In some aspects, the vector is a plasmid vector. In other aspects, the vector is a viral vector. In some examples, the viral vector is an adenovirus vector, an AAV vector, or a lentivirus vector.

[0234] V. Methods for Editing the ALPK1 Gene

[0235] The present disclosure further provides methods for correcting the C.710OT mutation in the human ALPK1 gene using a particular gRNA sequence and ABE combination that results in highly efficient correction of the C710T mutation in the ALPK1 gene with little to no off-target / bystander effects. 4239-112678-02

[0236] A. Methods of Editing the ALPK1 Gene in an Isolated Cell

[0237] Provided herein are methods of editing a thymine mutation at nucleotide 710 of the human ALPK1 gene in an isolated cell by administering to the isolated cell (i) a gRNA targeting the ALPK1 gene, or a nucleic acid molecule encoding the gRNA; and (ii) a modified ABE, or a nucleic acid molecule encoding the modified ABE.

[0238] In some aspects, the nucleotide sequence of the gRNA includes SEQ ID NO: 1 with no more than 4, no more than 3, no more than 2, or no more than 1 nucleotide substitution(s). In some examples, the nucleotide sequence of the gRNA includes SEQ ID NO: 1 and up to 100, up to 90, up to 80, up to 70, up to 60, up to 50, up to 40, up to 30, up to 20, or up to 10 additional nucleotide(s) at the 5' end of SEQ ID NO: 1 and / or up to 100, up to 90, up to 80, up to 70, up to 60, up to 50, up to 40, up to 30, up to 20, or up to 10 additional nucleotide(s) at the 3' end of SEQ ID NO: 1. In specific examples, the total length of the gRNA is about 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 or 120 nucleotides in length, such as about 90-110, about 95-105, or about 97-103 nucleotides in length. In one non-limiting example, the gRNA sequence is 100 nucleotides in length.

[0239] In some aspects, the modified ABE incorporates the R691 A HiFi mutation previously described for Cas9 (Vakulskas et al., Nat Med 24(8): 1216-1224, 2018). In some examples, the amino acid sequence of the modified ABE includes or consists of SEQ ID NO: 14 with no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid substitution(s), such as conservative amino acid substitution(s), while retaining the HiFi mutation (in SEQ ID NO: 14, the HiFi mutation is located at residue 1105). In specific examples, the amino acid sequence of the modified ABE includes or consists of SEQ ID NO: 14.

[0240] In some aspects, the nucleic acid molecule encoding the modified base editor is a DNA molecule. In other aspects, the nucleic acid molecule encoding the modified base editor is a mRNA molecule. In some aspects, the nucleotide sequence encoding the modified ABE is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 15. In some examples, the nucleotide sequence encoding the modified ABE includes or consists of SEQ ID NO: 15.

[0241] In some aspects, the gRNA and the base editor are administered as a ribonucleoprotein (RNP) complex. In some examples, the RNP complex is administered to the isolated cell by electroporation, microinjection or lipid nanoparticle. 4239-112678-02

[0242] In some aspects, the gRNA and the nucleic acid molecule encoding the base editor are administered in a lipid nanoparticle. In other aspects, the nucleic acid molecule encoding the gRNA and the nucleic acid molecule encoding the base editor are administered in a lipid nanoparticle.

[0243] In some aspects, the nucleic acid molecule encoding the gRNA and the nucleic acid molecule encoding the base editor are administered in a viral vector. In some examples, the viral vector is an AAV vector, a lentivirus vector, or an adenovirus vector.

[0244] In some aspects of the methods, the isolated cell is an induced pluripotent stem cell (iPSC). In some examples, the iPSC is produced from a cell obtained from a subject harboring a cytosine to thymine mutation at nucleotide 710 of the human ALPK1 gene. In some examples, the edited iPSC is differentiated to a relevant cell type (such as retinal pigmented epithelium) and the differentiated cell is administered to the subject harboring the ALPK1 mutation.

[0245] In other aspects, the isolated cell is a hematopoietic stem cell. In some examples, the hematopoietic stem cell is obtained from a subject harboring a cytosine to thymine mutation at nucleotide 710 of the human ALPK1 gene. In some examples, the edited hematopoietic stem cell is administered to a subject harboring the ALPK1 mutation for use in bone marrow transplantation.

[0246] In some aspects of the method, the rate of correction of the mutation in ALPK1 is at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. The rate of correction can be determined by nucleic acid sequencing before and after treatment using known methods.

[0247] B. Methods of Editing the ALPK1 Gene in a Subject

[0248] Further provided herein are methods of correcting a genetic mutation in a subject, wherein the genetic mutation is a cytosine to thymine mutation at nucleotide 710 of the human ALPK1 gene. The methods include administering to the subject (i) a gRNA targeting the ALPK1 gene, or a nucleic acid molecule encoding the gRNA; and (ii) a modified ABE, or a nucleic acid molecule encoding the modified ABE.

[0249] In some aspects, the subject has ROSAH syndrome. In other aspects, the subject has a tumor with a somatic mutation in the ALPK1 gene.

[0250] In some aspects, the nucleotide sequence of the gRNA includes SEQ ID NO: 1 with no more than 4, no more than 3, no more than 2, or no more than 1 nucleotide substitution(s). 4239-112678-02

[0251] In some examples, the nucleotide sequence of the gRNA includes SEQ ID NO: 1 and up to 100, up to 90, up to 80, up to 70, up to 60, up to 50, up to 40, up to 30, up to 20, or up to 10 additional nucleotide(s) at the 5’ end of SEQ ID NO: 1 and / or up to 100, up to 90, up to 80, up to 70, up to 60, up to 50, up to 40, up to 30, up to 20, or up to 10 additional nucleotide(s) at the 3' end of SEQ ID NO: 1. In specific examples, the total length of the gRNA is about 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 or 120 nucleotides in length, such as about 90-110, about 95-105, or about 97-103 nucleotides in length. In one non-limiting example, the gRNA sequence is 100 nucleotides in length.

[0252] In some aspects, the modified ABE incorporates the R691 A HiFi mutation previously described for Cas9 (Vakulskas et al., Nat Med 24(8): 1216-1224, 2018). In some examples, the amino acid sequence of the modified ABE includes or consists of SEQ ID NO: 14 with no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid substitution(s), such as conservative amino acid substitution(s), while retaining the HiFi mutation (in SEQ ID NO: 14, the HiFi mutation is located at residue 1105). In specific examples, the amino acid sequence of the modified ABE includes or consists of SEQ ID NO: 14.

[0253] In some aspects, the nucleic acid molecule encoding the modified base editor is a DNA molecule. In other aspects, the nucleic acid molecule encoding the modified base editor is a mRNA molecule. In some aspects, the nucleotide sequence encoding the modified ABE is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 15. In some examples, the nucleotide sequence encoding the modified ABE includes or consists of SEQ ID NO: 15.

[0254] In some aspects, the gRNA and the base editor are administered as a RNP complex.

[0255] In some aspects, the gRNA and the nucleic acid molecule encoding the base editor are administered in a lipid nanoparticle. In other aspects, the nucleic acid molecule encoding the gRNA and the nucleic acid molecule encoding the base editor are administered in a lipid nanoparticle.

[0256] In some aspects, the nucleic acid molecule encoding the gRNA and the nucleic acid molecule encoding the base editor are administered in a viral vector. In some examples, the viral vector is an AAV vector, a lentivirus vector, or an adenovirus vector.

[0257] In some aspects, administration (of the gRNA and base editor, or nucleic acid molecules encoding the gRNA and the base editor) comprises intravenous administration, 4239-112678-02 intraocular administration, portal vein administration, intratumoral administration, or retroductal cannulation of a salivary gland.

[0258] In some aspects of the method, the rate of correction of the mutation in ALPK1 is at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. The rate of correction can be determined by nucleic acid sequencing before and after treatment using known methods.

[0259] Editing of the ALPK 1 gene can be performed to treat a number of different symptoms (such as any symptom related to ROSAH syndrome) or disease states (such as tumors expressing mutant ALPK1). In some instances, a subject has retinal degeneration (such as retinal degeneration resulting from ROSAH syndrome) and the subject is treated using the gene editing methods disclosed herein. For example, the subject can be administered the gRNA and base editor (or nucleic acid molecules / vectors encoding the gRNA and base editor) intraocularly, such as by intravitreal administration. In other instances, the subject suffers from dry mouth resulting from the presence of the ALPK1 mutation in the salivary glands. Such subjects can be treated using the gene editing methods disclosed herein, such as by administration of the gRNA and base editor (or nucleic acid molecules / vectors encoding same) via retroductal cannulation of a salivary gland. If delivery to the liver of a subject is desired, administration of the gRNA and base editor (or nucleic acid molecules / vectors encoding same) can be intravenous or via portal vein delivery. Some subjects with ROSAH syndrome are more susceptible to the development of tumors. Thus, the gene editing methods disclosed herein can be administered to a subject to inhibit growth of a tumor, or can be administered prophylactically to prevent development of a tumor driven by the ALPK1 mutation (for example, to prevent salivary gland tumors). In some examples, the subject with a tumor (or at risk of a tumor) is administered the gRNA and base editor (or nucleic acid molecules / vectors encoding same) intratumorally, intravenously, or by retroductal cannulation of a salivary gland.

[0260] C. Ex Vivo Methods of Editing the ALPK1 Gene

[0261] Also provided herein are ex vivo methods of correcting a genetic mutation, wherein the genetic mutation is a cytosine to thymine mutation at nucleotide 710 of the human ALPK1 gene. In such methods, induced pluripotent stem cells (iPSCs) are contacted with a modified base editor and gRNA disclosed herein to correct the ALPK1 gene ex vivo, after 4239-112678-02 which the iPSCs are differentiated to an appropriate cell type and then administered to a subject in need of ALPK1 gene editing, such as a subject with ROSAH syndrome.

[0262] In some aspects, the method includes (i) administering to an iPSC a gRNA targeting the ALPK1 gene, or a nucleic acid molecule encoding the gRNA; and a modified ABE, or a nucleic acid molecule encoding the modified ABE, thereby correcting the ALPK1 gene in the iPSC; (ii) differentiating the iPSC with the corrected ALPK1 gene to a clinically relevant cell type (e.g., retinal pigmented epithelium (RPE) or photoreceptor cells); and (iii) administering the differentiated cells to a subject. In some examples, the subject has ROSAH syndrome. In some examples, the subject has retinal degeneration.

[0263] In some aspects, the iPSC is produced from a cell obtained from a subject harboring a cytosine to thymine mutation at nucleotide 710 of the human ALPK1 gene. In some examples, the method further includes, prior to step (i), isolating cells from the subject harboring a cytosine to thymine mutation at nucleotide 710 of the human ALPK1 gene, such as a subject with ROSAH syndrome, and reprogramming the cells into iPSCs, such as under clinical grade conditions. Methods for producing clinical grade iPSCs and reprogramming the iPSCs into retinal pigmented epithelium are described in, for example, Sharma et al. (Sci Transl Med l l(475):eaat5580, 2019).

[0264] In some aspects, following step (ii), the differentiated cells are cultured on a biodegradable scaffold or other support material that enables formation of a polarized, tissuelike structure (e.g., a polarized monolayer). As the scaffold degrades, the cells establish their own extracellular matrix and develop an organized architecture compatible with transplantation. For an exemplary method, see Gupta et al. (J CI Insight 10(10):el79246, 2025).

[0265] In some aspects, the nucleotide sequence of the gRNA includes SEQ ID NO: 1 with no more than 4, no more than 3, no more than 2, or no more than 1 nucleotide substitution(s). In some examples, the nucleotide sequence of the gRNA includes SEQ ID NO: 1 and up to 100, up to 90, up to 80, up to 70, up to 60, up to 50, up to 40, up to 30, up to 20, or up to 10 additional nucleotide(s) at the 5' end of SEQ ID NO: 1 and / or up to 100, up to 90, up to 80, up to 70, up to 60, up to 50, up to 40, up to 30, up to 20, or up to 10 additional nucleotide(s) at the 3' end of SEQ ID NO: 1. In specific examples, the total length of the gRNA is about 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 or 120 nucleotides in length, such as about 90-110, about 95-105, or about 97-103 nucleotides in length. In one non-limiting example, the gRNA sequence is 100 nucleotides in length. 4239-112678-02

[0266] In some aspects, the modified ABE incorporates the R691A HiFi mutation previously described for Cas9 (Vakulskas et al., Nat Med 24(8): 1216-1224, 2018). In some examples, the amino acid sequence of the modified ABE includes or consists of SEQ ID NO: 14 with no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid substitution(s), such as conservative amino acid substitution(s), while retaining the HiFi mutation (in SEQ ID NO: 14, the HiFi mutation is located at residue 1105). In specific examples, the amino acid sequence of the modified ABE includes or consists of SEQ ID NO: 14.

[0267] In some aspects, the nucleic acid molecule encoding the modified base editor is a DNA molecule. In other aspects, the nucleic acid molecule encoding the modified base editor is a mRNA molecule. In some aspects, the nucleotide sequence encoding the modified ABE is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 15. In some examples, the nucleotide sequence encoding the modified ABE includes or consists of SEQ ID NO: 15.

[0268] In some aspects, the gRNA and the base editor are administered as a ribonucleoprotein (RNP) complex. In some examples, the RNP complex is administered to the iPSC by electroporation, microinjection or lipid nanoparticle.

[0269] In some aspects, the gRNA and the nucleic acid molecule encoding the base editor are administered in a lipid nanoparticle. In other aspects, the nucleic acid molecule encoding the gRNA and the nucleic acid molecule encoding the base editor are administered in a lipid nanoparticle.

[0270] In some aspects, the nucleic acid molecule encoding the gRNA and the nucleic acid molecule encoding the base editor are administered in a viral vector. In some examples, the viral vector is an AAV vector, a lentivirus vector, or an adenovirus vector.

[0271] In some aspects of the method, the rate of correction of the mutation in ALPK1 is at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. The rate of correction can be determined by nucleic acid sequencing before and after treatment using known methods. 4239-112678-02

[0272] EXAMPLES

[0273] The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified.

[0274] Example 1: Screening of guide RNAs targeting the ALPK1 c.710C>T (Thr237Met) mutation for base editor correction

[0275] This example compares gene editing capability of the adenine base editor ABE7. 10- SpRY using either the A5 gRNA (SEQ ID NO: 1) or the A4 gRNA (SEQ ID NO: 2).

[0276] Induced pluripotent stem cells (iPSCs) were produced from a patient with ROS AH syndrome who is heterozygous for the ALPK1 C.710OT mutation. The iPSCs were either unedited, or edited using the A5 gRNA or the A4 gRNA. The A5 gRNA targets the DNA sequence CGACATGGAGAGGCCCTGTA (SEQ ID NO: 1), with the underlined “A” in position 5 of the gRNA sequence corresponding to the patient C>T mutation on the reverse complementary DNA strand. This gRNA has a protospacer-adjacent motif (PAM) sequence of “AGA” rather than the canonical SpCas9 “NGG” PAM, thereby requiring a SpRY “P AM- less” version of the base editor to target this sequence. The A4 gRNA targets the DNA sequence GACATGGAGAGGCCCTGTAA (SEQ ID NO: 2), with the underlined “A” in position 4 of the gRNA sequence corresponding to the patient C>T mutation on the reverse complementary DNA strand. This guide RNA also requires a SpRY “PAM-less” base editor to target this sequence due to its non-canonical PAM.

[0277] As shown in FIG. 1A, the unedited iPSCs show 50% C and 50% T at residue 710 of the ALPK1 gene. Sanger sequencing of the A5-edited iPSCs revealed approximately 100% correction of the patient mutation (FIG. IB), as indicated by complete conversion of the -50% C / T to 100% C in the c.710 position of the sequencing data. In addition, there was no bystander editing of nucleotides near the c.710 position. Sanger sequencing of the A4-edited iPSCs revealed poor correction of the patient mutation at position c.710, as well as a high degree of bystander editing at the nearby c.706 position (FIG. 1C), which would unintentionally induce a novel mutation. These data demonstrate that the A5 gRNA resulted in the highest rate of correction without bystander edits in the ALPK1 gene.

[0278] Example 2: Screening of alternative adenine base editors for correcting the ALPK1 c.710C>T (Thr237Met) mutation 4239-112678-02

[0279] This example compares the base editing capability of base editors ABE7.10-SpRY, ABE8e-HiFi-SpRY and ABE9-SpRY using the A5 gRNA. iPSCs were produced from a patient with ROS AH syndrome who is heterozygous for the ALPK1 C.710OT mutation. The iPSCs were either unedited, or edited using the A5 gRNA and a base editor selected from ABE7.10-SpRY, ABE8e-HiFi-SpRY and ABE9- SpRY.

[0280] As shown in FIG. 2A, the unedited iPSCs show 50% C and 50% T at residue 710 of the ALPK1 gene. Editing of patient iPSCs with ABE7. 10-SpRY and the A5 gRNA led to approximately 100% correction without bystander edits (FIG. 2B). As shown in FIG. 2C, editing of patient iPSCs with ABE8e-HiFi-SpRY and the A5 gRNA resulted in approximately 100% correction, but caused a high degree of bystander editing at the nearby c.706 (the same bystander edit caused by the A4 gRNA with base editor ABE7.10-SpRY). Editing of patient iPSCs with the A5 gRNA and ABE9-SpRY (an alternative base editor having a narrower editing window than ABE7.10 or ABE8e, for reduced bystander editing) resulted in no bystander edits, but poor correction of the c.710 mutation (FIG. 2D).

[0281] These results demonstrate that the ABE7.10-SpRY base editor produces the highest rate of correction without bystander edits in the ALPK1 gene.

[0282] Example 3: Whole genome sequencing of edited patient iPSCs reveal a potent off-target edit with ABE7.10-SpRY and A5 gRNA

[0283] Whole genome sequencing of two biological replicates of patient iPSCs edited with AB E7.10-SpRY and A5 guide RNA revealed a number of off-target edits occurring either in inter-gene regions (IGRs), intronic regions within genes, downstream untranslated regions (3' UTRs) or upstream flanking regions of genes (5' Flank), as well as a missense mutation within the FGFR2 gene that occurred with a high frequency in both edited iPSC samples. The off-target mutation was detected in 14 out of 29 sequencing reads for edited sample #1 (947Dpolyclnl), and in 13 out of 37 reads for edited sample #2 (947Dpolyclnl), and a neighboring silent mutation in the same region of the FGFR2 gene. These FGFR2 mutations occurred within a region with 17 out of 20 matching nucleotides to the ALPK1 sequence targeted by the A5 gRNA, confirming that these were off-target edits caused by the combination of ABE7.10-SpRY and A5 gRNA. This data is summarized in FIGS. 3A-3B.

[0284] These results demonstrated that the combination of the AB E7.10-SpRY base editor and the A5 guide RNA caused a potentially risky off-target edit in the FGFR2 gene. 4239-112678-02

[0285] Example 4: Sanger sequencing of edited patient iPSCs at the FGFR2 off-target site for different adenine base editors in combination with the A5 gRNA

[0286] This example compares base editing of the FGFR2 off-target mutation using the A5 gRNA and base editor ABE7.10-SpRY, ABE8e-HiFi-SpRY or ABE9-SpRY. iPSCs were produced from a patient with ROSAH syndrome who is heterozygous for the ALPK1 C.710OT mutation and does not contain mutations at positions c.1681 and c.1683 of the FGFR2 gene. The iPSCs were either unedited, or edited using the A5 gRNA and a base editor selected from ABE7.10-SpRY, ABE8e-HiFi-SpRY and ABE9-SpRY.

[0287] As shown in FIG. 4A, unedited patient iPSCs lack the FGFR2 off-target mutation (100% T at positions 1681 and 1683). As expected based on whole genome sequencing, patient iPSCs edited with ABE7.10-SpRY and the A5 gRNA showed both the FGFR2 off- target silent (c.1683) and missense (c.1681) mutations (FIG. 4B). Patient iPSCs edited with ABE8e-HiFi-SpRY and the A5 gRNA showed even higher levels of the off-target silent and missense FGFR2 mutations (FIG. 4C). As shown in FIG. 4D, patient iPSCs edited with ABE9-SpRY and the A5 gRNA showed lower levels of the off-target FGFR2 missense mutation and none the off-target FGFR2 silent mutation. However, the ABE9-SpRY base editor also led to lower on-target ALPK1 editing (see FIG. 2D).

[0288] These data show that all three adenine base editors tested cause a potentially risky off- target edit in the FGFR2 gene.

[0289] Example 5: Comparison of ABE7.10-SpRY and high-fidelity ABE7.10-HiFi-SpRY base editors for the FGFR2 off-target edits in edited patient iPSCs

[0290] A previously described high-fidelity (“HiFi”) mutation in Cas9 (R691 A) was reported to confer reduced off-target editing (Vakulskas et al., Nat Med 24(8): 1216- 1224, 2018). However, this mutation had not been previously tested in the ABEmax(7. 10)-SpRY base editor. To determine whether the HiFi mutation could reduce off target effects observed with ABEmax(7.10)-SpRY, the base editor was modified to incorporate this mutation, resulting in ABE7. 10-HiFi-SpRY (SEQ ID NO: 14). A map of the plasmid encoding the modified ABE7. 10-HiFi-SpRY base editor is shown in FIG. 5. iPSCs were produced from two different patients with ROSAH syndrome who are heterozygous for the ALPK1 C.710OT mutation and do not contain mutations at positions c.1681 and c.1683 of the FGFR2 gene. The iPSCs were either unedited or edited using the A5 gRNA and ABEmax(7. 10)-SpRY or ABE7. 10-HiFi-SpRY. 4239-112678-02

[0291] As shown in FIGS. 6A-6B. Sanger sequencing of the FGFR2 off-target site in unedited iPSCs from two different ROSAH patients confirms that the unedited iPSCs do not have the off-target mutations (100% T at c.1681 and c.1683). Sanger sequencing data of iPSCs from one of the ROSAH patients edited with the regular-fidelity ABE7.10-SpRY base editor and A5 gRNA shows the previously discovered FGFR2 off-target edits at c.1681 and c.1683 (FIG. 6C). As shown in FIGS. 6D-6F, three replicates of patient iPSCs (two replicates for iPSCs from patient #1 and one replicate for iPSCs from patient #2) edited with the new high-fidelity ABE7. 10-HiFi-SpRY base editor and A5 guide RNA demonstrated no FGFR2 off-target edits.

[0292] These data demonstrate that the high-fidelity AB E7.10-HiFi-SpRY negates the FGFR2 off-target editing problem.

[0293] Example 6: Comparison of the regular fidelity ABE7.10-SpRY base editor with the modified high-fidelity ABE7.10-HiFi-SpRY base editor for the on-target ALPK1 c.710 correction in edited patient iPSCs iPSCs were produced from two different patients with ROSAH syndrome who are heterozygous for the ALPK1 c.710C>T mutation. The iPSCs were either unedited or edited using the A5 gRNA and the ABEmax(7.10)-SpRY or the ABE7.10-HiFi-SpRY base editor.

[0294] As shown in FIGS. 7A-7B, Sanger sequencing confirmed the presence of the ALPK1 c.710C>T mutation in iPSCs from two different ROSAH patients. Sanger sequencing data of iPSCs from one of the ROSAH patients edited with the regular-fidelity ABE7.10-SpRY base editor and A5 gRNA showed approximately 100% correction (FIG. 7C). As shown in FIGS. 7D-7F, three replicates of patient iPSCs (two replicates for iPSCs from patient #1 and one replicate for iPSCs from patient #2) edited with the high-fidelity AB E7.10-HiFi-SpRY base editor and A5 gRNA demonstrated significant levels on-target correction (-68-76% correction efficiency) without bystander edits, based on the detection limitations of Sanger sequencing.

[0295] These data demonstrate that the AB E7.10-HiFi-SpRY base editor (in combination with A5 gRNA) corrects the ALPK1 mutation with a modest reduction in efficiency compared to ABEmax(7. 10)-SpRY, but has the advantage of no off-target edits. 4239-112678-02

[0296] Example 7: Whole genome sequencing data for on- target ALPK1 c.710 editing in patient iPSCs edited with A5 gRNA and the regular fidelity ABE7.10-SpRY or the high- fidelity ABE7.10-HiFi-SpRY base editor

[0297] Whole genome sequencing incorporates a greater number of DNA sequencing reads (~40 reads per cell sample) than Sanger sequencing (1 read per cell sample), for a more sensitive and in-depth assessment of correction efficiency. Based on whole genome sequencing data, the level of correction of the ALPK1 mutation in the three replicate iPSC correction samples was 68-94% (compared to the -68-76% correction detected for these three samples by Sanger sequencing in FIGS. 7D-7F). These results are summarized in FIG. 8.

[0298] It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

4239-112678-02CLAIMS1. A modified adenine base editor (ABE), wherein the amino acid sequence of the modified ABE comprises or consists of SEQ ID NO: 14.

2. A nucleic acid molecule encoding the modified ABE of claim 1.

3. The nucleic acid molecule of claim 2, which is codon-optimized for expression in human cells.

4. The nucleic acid molecule of claim 2 or claim 3, operably linked to a promoter.

5. A vector comprising the nucleic acid molecule of any one of claims 2-4.

6. The vector of claim 5, which is a plasmid vector.

7. The vector of claim 5, which is a viral vector.

8. The vector of claim 7, wherein the viral vector is an adeno-associated virus(AAV) vector, an adenovirus vector, or a lentivirus vector.

9. The vector of any one of claims 5-8, further comprising a nucleic acid sequence encoding a guide RNA (gRNA).

10. The vector of claim 9, wherein the nucleotide sequence of the gRNA comprises SEQ ID NO: 1.

11. A guide RNA (gRNA) targeting the human ALPK 1 gene, wherein the nucleotide sequence of the gRNA comprises SEQ ID NO: 1.

12. A method of editing a thymine mutation at nucleotide 710 of the human alpha kinase 1 (ALPK1) gene in an isolated cell, comprising administering to the isolated cell:4239-112678-02 a guide RNA (gRNA) comprising the nucleotide sequence of SEQ ID NO: 1, or a nucleic acid molecule encoding a gRNA comprising the nucleotide sequence of SEQ ID NO: 1; and a modified base editor comprising the amino acid sequence of SEQ ID NO: 14, or a nucleic acid molecule encoding the modified base editor.

13. The method of claim 12, wherein the nucleotide sequence of the gRNA comprises SEQ ID NO: 1 and up to about 80 additional nucleotides.

14. The method of claim 12 or claim 13, wherein the amino acid sequence of the modified base editor consists of SEQ ID NO: 14.

15. The method of any one of claims 12-14, wherein the nucleic acid molecule encoding the modified base editor is a DNA molecule.

16. The method of any one of claims 12-14, wherein the nucleic acid molecule encoding the modified base editor is a mRNA molecule.

17. The method of any one of claims 12-16, wherein the gRNA and the base editor are administered as a ribonucleoprotein (RNP) complex.

18. The method of claim 17, wherein the RNP complex is administered to the isolated cell by electroporation, microinjection or lipid nanoparticle.

19. The method of any one of claims 12-16, wherein: the gRNA and the nucleic acid molecule encoding the base editor are administered in a lipid nanoparticle; or the nucleic acid molecule encoding the gRNA and the nucleic acid molecule encoding the base editor are administered in a lipid nanoparticle.

20. The method of any one of claims 12-16, wherein the nucleic acid molecule encoding the gRNA and the nucleic acid molecule encoding the base editor are administered in a viral vector.4239-112678-0221. The method of claim 20, wherein the viral vector is an adeno-associated virus (AAV) vector, a lenti virus vector, or an adenovirus vector.

22. The method of any one of claims 12-21, wherein the isolated cell is an induced pluripotent stem cell (iPSC) or a hematopoietic stem cell.

23. The method of claim 22, wherein the iPSC is produced from a cell obtained from a subject harboring a cytosine to thymine mutation at nucleotide 710 of the human ALPK1 gene.

24. A method of correcting a genetic mutation in a subject, wherein the genetic mutation is a cytosine to thymine mutation at nucleotide 710 of the human ALPK1 gene, comprising administering to the subject: a guide RNA (gRNA) comprising the nucleotide sequence of SEQ ID NO: 1, or a nucleic acid molecule encoding a gRNA comprising the nucleotide sequence of SEQ ID NO: 1; and a modified base editor comprising the amino acid sequence of SEQ ID NO: 14, or a nucleic acid molecule encoding the modified base editor.

25. The method of claim 24, wherein the subject has ROSAH syndrome or a tumor with a somatic mutation in the ALPK1 gene.

26. The method of claim 24 or claim 25, wherein the nucleotide sequence of the gRNA comprises SEQ ID NO: 1 and up to about 80 additional nucleotides.

27. The method of any one of claims 24-26, wherein the amino acid sequence of the modified base editor consists of SEQ ID NO: 14.

28. The method of any one of claims 24-27, wherein the nucleic acid molecule encoding the modified base editor is DNA.

29. The method of any one of claims 24-27, wherein the nucleic acid molecule encoding the modified base editor is mRNA.4239-112678-0230. The method of any one of claims 24-29, wherein the gRNA and the base editor are administered as a ribonucleoprotein (RNP) complex.

31. The method of claim 30, wherein the RNP complex is administered in a lipid nanoparticle.

32. The method of any one of claims 24-29, wherein: the gRNA and the nucleic acid molecule encoding the base editor are administered in a lipid nanoparticle; or the nucleic acid molecule encoding the gRNA and the nucleic acid molecule encoding the base editor are administered in a lipid nanoparticle.

33. The method of any one of claims 24-29, wherein the nucleic acid molecule encoding the gRNA and the nucleic acid molecule encoding the base editor are administered in a viral vector.

34. The method of claim 33, wherein the viral vector is an adeno-associated virus (AAV) vector, a lentivirus vector, or an adenovirus vector.

35. The method of any one of claims 24-34, wherein administration comprises intravenous administration, intraocular administration, portal vein administration, intratumoral administration, or retroductal cannulation of a salivary gland.

36. An ex vivo method of editing a thymine mutation at nucleotide 710 of the human alpha kinase 1 (ALPK1) gene in induced pluripotent stem cells (iPSCs), comprising:(i) administering to the iPSCs a guide RNA (gRNA) comprising the nucleotide sequence of SEQ ID NO: 1, or a nucleic acid molecule encoding a gRNA comprising the nucleotide sequence of SEQ ID NO: 1; and a modified base editor comprising the amino acid sequence of SEQ ID NO: 14, or a nucleic acid molecule encoding the modified base editor;(ii) differentiating the iPSCs to differentiated cells; and(iii) administering the differentiated cells to a subject harboring a cytosine to thymine mutation at nucleotide 710 of the human ALPK1 gene.4239-112678-0237. The ex vivo method of claim 36, further comprising, prior to step (i), isolating cells from the subject harboring a cytosine to thymine mutation at nucleotide 710 of the human ALPK1 gene and reprogramming the cells into iPSCs.

38. The method of claim 36 or claim 37, wherein the iPSCs are differentiated to retinal pigmented epithelial cells.

39. The method of claim 38, wherein the differentiated retinal pigmented epithelial cells are cultured on a biodegradable scaffold to form a polarized monolayer.

40. The method of any one of claims 36-39, wherein the subject has ROSAH syndrome.

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