IP Library › Patent Application 17929828

Patent Application

App. No. 17/929,828

COMPOSITIONS AND METHODS FOR MODULATING FORKHEAD BOX P3 (FOXP3) GENE EXPRESSION

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Patent No.: US None
App. No.: 17/929,828

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Abstract

The present invention provides agents and compositions for modulating expression (e.g., enhanced or reduced expression) of a forkhead box P3 (FOXP3) gene by targeting a FOXP3 expression control region and methods of use thereof for treating a FOXP3 associated disorder, such as an autoimmune disease, e.g., IPEX syndrome.

Claims (160)

1 . A site-specific forkhead box P3(FOXP3) disrupting agent, comprising a site-specific FOXP3 targeting moiety which targets a FOXP3 expression control region.

2 . The site-specific FOXP3 disrupting agent of claim 1 ,

(a) wherein the site-specific FOXP3 targeting moiety comprises a polymeric molecule; optionally a polyamide or a polynucleotide; optionally wherein the polymeric molecule comprises a peptide nucleic acid (PNA);

(b) wherein the expression control region comprises a region upstream of a FOXP3 transcription start site (TSS);

(c) wherein the expression control region comprises one or more FOXP3-associated anchor sequences within an anchor sequence-mediated conjunction comprising a first and a second FOXP3-associated anchor sequence;

optionally, wherein the anchor sequence comprises a CCCTC-binding factor (CTCF) binding motif;

optionally, wherein the anchor sequence-mediated conjunction comprises one or more transcriptional control elements internal to the conjunction, or one or more transcriptional control elements external to the conjunction;

optionally, wherein the first and/or the second anchor sequence is located within about 500 kb, or within about 300 kb, or within about 10 kb, of the transcriptional control element;

(d) wherein the expression control region comprises a FOXP3-specific transcriptional control element, optionally, wherein the transcriptional control element comprises a FOXP3 promoter, a transcriptional enhancer, or a transcriptional repressor; and/or

(e) wherein the disrupting agent comprises a modification.

3 - 16 . (canceled)

17 . The site-specific FOXP3 disrupting agent of claim 1 , wherein the FOXP3 targeting moiety comprises

(a) a nucleotide sequence having at least 85% nucleotide identity to the entire nucleotide sequence of any one of the nucleotide sequences in Table 2;

(b) a first nucleotide sequence having at least 85% nucleotide identity to the entire nucleotide sequence of GD-28448, a second nucleotide sequence having at least 85% nucleotide identity to the entire nucleotide sequence of GD-28449, and a third nucleotide sequence having at least 85% nucleotide identity to the entire nucleotide sequence of GD-28450; and/or

(c) a polymeric molecule comprises a polynucleotide encoding a DNA-binding domain, or fragment thereof, of a zinc finger polypeptide (ZNF) or a transcription activator-like effector (TALE) polypeptide that specifically binds to the FOXP3 expression control region; optionally, wherein the DNA-binding domain of the TALE or ZNF polypeptide comprises an amino acid sequence having at least about 85% amino acid identity to the entire amino acid sequence of any one of the amino acid sequences listed in Table 1B.

18 - 22 . (canceled)

23 . A vector comprising the site-specific FOXP3 disrupting agent of claim 1 , optionally, wherein the vector is a viral expression vector.

24 . (canceled)

25 . A cell comprising the site-specific FOXP3 disrupting agent of claim 1 , optionally, wherein the cell is an immune cell.

26 . (canceled)

27 . (canceled)

28 . The site-specific FOXP3 disrupting agent of claim 1 , wherein the site-specific FOXP3 disrupting agent is present in a composition; optionally, wherein the composition comprises a pharmaceutical composition; optionally,

(a) wherein the pharmaceutical composition comprises a lipid formulation, wherein the lipid formulation comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, or one or more PEG-modified lipids, or combinations of any of the foregoing; and/or

(b) wherein the pharmaceutical composition comprises a lipid nanoparticle.

29 - 32 . (canceled)

33 . A site-specific FOXP3 disrupting agent, comprising a nucleic acid molecule encoding a fusion protein, the fusion protein comprising a site-specific FOXP3 targeting moiety which targets a FOXP3 expression control region and an effector molecule.

34 . The site-specific FOXP3 disrupting agent of claim 33 ,

(a) wherein the site-specific FOXP3 targeting moiety comprises a polynucleotide encoding a DNA-binding domain, or fragment thereof, of a zinc finger polypeptide (ZNF) or a transcription activator-like effector (TALE) polypeptide that specifically binds to the FOXP3 expression control region; optionally, wherein the DNA-binding domain of the TALE or zinc finger polypeptide comprises an amino acid sequence having at least 85% amino acid identity to the entire amino acid sequence of an amino acid sequence selected from the amino acid sequences listed in Table 1B;

(b) wherein the effector molecule comprises a nucleic acid molecule encoding a polypeptide;

(d) wherein the effector is selected from the group consisting of a nuclease, a physical blocker, an epigenetic recruiter, and an epigenetic CpG modifier, and combinations of any of the foregoing;

(e) wherein the effector comprises a CRISPR associated protein (Cas) polypeptide or nucleic acid molecule encoding the Cas polypeptide; optionally, wherein the Cas polypeptide is an enzymatically inactive Cas polypeptide, or further comprises a catalytically active domain of human exonuclease 1 (hEXO1);

(f) wherein the epigenetic recruiter comprises a transcriptional enhancer or a transcriptional repressor;

(g) wherein the epigenetic CpG modifier comprises a DNA methylase, a DNA demethylase, a histone modifying agent, a histone transacetylase, or a histone deacetylase;

(h) wherein the effector molecule comprises a zinc finger polypeptide; and/or

(i) wherein the effector molecule comprises a Transcription activator-like effector nuclease (TALEN) polypeptide.

35 - 42 . (canceled)

43 . The site-specific FOXP3 disrupting agent of claim 34 ,

(a) wherein the transcriptional enhancer is a VPR (VP64-p65-Rta); optionally,

wherein the VPR comprises an amino acid sequence having at least about 85% amino acid identity to the entire amino acid sequence of DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLSGGPKKKRKVGSQY LPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTIN YDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAP KPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPML MEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLGSGSGSRDSREGMFL PKPEAGSAISDVFEGREVCQPKRLRPFHPPGSPWANRPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDP APAVTPEASHLLEDPDEETSQAVKALREMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESM TEDLNLDSPLTPELNEILDTFLNDECLLHAMHISTGLSIFDTSLF (SEQ ID NO: 64); and/or,

optionally, wherein the transcriptional enhancer comprises two, three, four, or five VPRs; and/or

(b) wherein the transcriptional enhancer is a p300; optionally,

wherein the p300 comprises an amino acid sequence having at least about 85% identity to the entire amino acid sequence of

(SEQ ID NO: 65)

IFKPEELRQALMPTLEALYRQDPESLPFRQPVDPQLLGIPDYFDIVKSPM

DLSTIKRKLDTGQYQEPWQYVDDIWLMFNNAWLYNRKTSRVYKYCSKLSE

VFEQEIDPVMQSLGYCCGRKLEFSPQTLCCYGKQLCTIPRDATYYSYQNR

YHFCEKCFNEIQGESVSLGDDPSQPQTTINKEQFSKRKNDTLDPELFVEC

TECGRKMHQICVLHHEIIWPAGFVCDGCLKKSARTRKENKFSAKRLPSTR

LGTFLENRVNDFLRRQNHPESGEVTVRVVHASDKTVEVKPGMKARFVDSG

EMAESFPYRTKALFAFEEIDGVDLCFFGMHVQEYGSDCPPPNQRRVYISY

LDSVHFFRPKCLRTAVYHEILIGYLEYVKKLGYTTGHIWACPPSEGDDYI

FHCHPPDQKIPKPKRLQEWYKKMLDKAVSERIVHDYKDIFKQATEDRLTS

AKELPYFEGDFWPNVLEESIKELEQEEEERKREENTSNESTDVTKGDSKN

AKKKNNKKTSKNKSSLSRGNKKKPGMPNVSNDLSQKLYATMEKHKEVFFV

IRLIAGPAANSLPPIVDPDPLIPCDLMDGRDAFLTLARDKHLEFSSLRRA

QWSTMCMLVELHTQSQD

44 - 50 . (canceled)

51 . The site-specific FOXP3 disrupting agent of claim 33 , further comprising a second nucleic acid molecule encoding a second fusion protein, wherein the second fusion comprises a second site-specific FOXP3 targeting moiety which targets a second FOXP3 expression control region and a second effector molecule, wherein the second FOXP3 expression control region is different than the FOXP3 expression control region; optionally,

(a) wherein the second effector is different than the effector;

(b) wherein the second effector is the same as the effector;

(d) wherein the fusion protein and the second fusion protein comprise an amino acid sequence that has at least about 85% amino acid sequence identity to the entire amino acid sequence of a polypeptide selected from the group consisting of dCas-P300 (SEQ ID NO: 10) and dCas-VPR (SEQ ID NO: 11); and/or

(e) wherein the fusion protein is encoded by a polynucleotide comprising a nucleotide sequence that has at least about 85% amino acid sequence identity to the entire nucleotide sequence of a polynucleotide selected from the group consisting of dCas-P300 mRNA (SEQ ID NO: 7) and dCas-VPR mRNA (SEQ ID NO: 8).

52 - 56 . (canceled)

57 . A site-specific FOXP3 disrupting agent, comprising

(a) a nucleic acid molecule encoding a fusion protein, wherein the fusion protein comprises an amino acid sequence having at least about 85% amino acid identity to the entire amino acid sequence of a polypeptide selected from the group consisting of dCas-P300 (SEQ ID NO: 10) and dCas-VPR (SEQ ID NO: 11;

(b) a polynucleotide encoding the amino acid sequence of dCas-P300

comprising the amino acid sequence of

(SEQ ID NO: 10)

MAPKKKRKVGIHGVPAADKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA

LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPI

FGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI

QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS

NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM

IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEE

LLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLAR

GNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNE

LTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNAS

LGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYT

GWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHI

ANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE

LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKV

LTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL

VETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDA

YLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN

GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR

KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYK

EVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQK

QLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAF

KYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDKRPAATKKAGQAKKKKGRAIFKP

EELRQALMPTLEALYRQDPESLPFRQPVDPQLLGIPDYFDIVKSPMDLSTIKRKLDTGQYQEPWQYV

DDIWLMFNNAWLYNRKTSRVYKYCSKLSEVFEQEIDPVMQSLGYCCGRKLEFSPQTLCCYGKQLC

TIPRDATYYSYQNRYHFCEKCFNEIQGESVSLGDDPSQPQTTINKEQFSKRKNDTLDPELFVECTECG

RKMHQICVLHHEIIWPAGFVCDGCLKKSARTRKENKFSAKRLPSTRLGTFLENRVNDFLRRQNHPES

GEVTVRVVHASDKTVEVKPGMKARFVDSGEMAESFPYRTKALFAFEEIDGVDLCFFGMHVQEYGS

DCPPPNQRRVYISYLDSVHFFRPKCLRTAVYHEILIGYLEYVKKLGYTTGHIWACPPSEGDDYIFHCH

PPDQKIPKPKRLQEWYKKMLDKAVSERIVHDYKDIFKQATEDRLTSAKELPYFEGDFWPNVLEESIK

ELEQEEEERKREENTSNESTDVTKGDSKNAKKKNNKKTSKNKSSLSRGNKKKPGMPNVSNDLSQK

LYATMEKHKEVFFVIRLIAGPAANSLPPIVDPDPLIPCDLMDGRDAFLTLARDKHLEFSSLRRAQWST

MCMLVELHTQSQDSGGKRPAATKKAGQAKKKKGSYPYDVPDYA;

and/or

comprising the amino acid sequence of

(SEQ ID NO: 11)