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US-20260124230-A1 - GENE EDITING FOR CONTROLLED EXPRESSION OF EPISOMAL GENES

US20260124230A1US 20260124230 A1US20260124230 A1US 20260124230A1US-20260124230-A1

Abstract

Disclosed herein are methods for regulating expression of a gene located on an episomal vector in a subject in need thereof. In particular, methods include administering to the subject one or more gene editing agents that modify a region of the gene to thereby regulate the expression of the gene. Also disclosed are methods of administering to the subject a base editor system that effects a base alteration in a region of the gene or a region of an mRNA transcript of the gene to thereby regulate the expression of the gene.

Inventors

  • SEAN ARMOUR
  • Ali Nahvi
  • Federico Mingozzi
  • Daniel Cohen
  • Yahui Lan

Assignees

  • SPARK THERAPEUTICS, INC.

Dates

Publication Date
20260507
Application Date
20231013

Claims (20)

  1. 1 . A method of regulating expression of a gene located on an episomal vector in a subject in need thereof, comprising administering to the subject an editing agent that effects an alteration in a region of an mRNA transcript of the gene to thereby regulate the expression of the gene.
  2. 2 . The method of claim 1 , wherein the editing agent effects a base alteration in the region of the mRNA transcript of the gene.
  3. 3 . The method of claim 1 , wherein the alteration in a region of an mRNA transcript of the gene alters the stability of the mRNA transcript, the initiation or level of the translation of the mRNA transcript, the stability and/or activity of the translated protein.
  4. 4 . The method of claim 2 , wherein the region of the mRNA transcript comprises a premature termination codon convertible to an amino acid codon via the base alteration, or an amino acid codon convertible to a premature termination codon via the base alteration.
  5. 5 . The method of claim 2 , wherein the base alteration (a) is within a microRNA targeting site or a toe-hold switch site, or (b) induces a ribosomal frameshift or alters a codon encoding an amino acid residue critical to function and/or structure of an encoded protein.
  6. 6 . The method of claim 2 , wherein the editing agent comprises a targeting ribonucleic acid complementary to the region of the mRNA transcript.
  7. 7 . The method of claim 6 , wherein the targeting ribonucleic acid is linear.
  8. 8 . The method of claim 6 , wherein the targeting ribonucleic acid is circular.
  9. 9 . The method of claim 6 , wherein the targeting ribonucleic acid effects the base alteration via binding to an endogenous adenosine deaminase domain.
  10. 10 . The method of claim 9 , wherein the adenosine deaminase is selected from the group consisting of adenosine deaminase acting on RNA 1 (ADAR1), ADAR2, and ADAR3.
  11. 11 . The method of claim 6 , wherein the editing agent further comprises: a base editor comprising a polynucleotide programmable RNA binding domain and an adenosine deaminase domain or a cytidine deaminase domain, or a nucleic acid encoding the based editor, wherein the polynucleotide programmable RNA binding domain in conjunction with the targeting ribonucleic acid effects the base alteration.
  12. 12 . The method of claim 11 , wherein the polynucleotide programmable RNA binding domain comprises a nuclease inactive variant of Cas13 or a nickase variant of Cas13.
  13. 13 . The method of claim 12 , wherein the Cas13 is Cas13a or Cas13b.
  14. 14 . The method of claim 11 , wherein (i) the cytidine deaminase domain is selected from the group consisting of the apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) family of deaminases, such as APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D/E, APOBEC3F, APOBEC3G, APOBEC3H, or APOBEC4; activation-induced cytidine deaminase (AID); cytosine deaminase 1 (CDA1) or CDA2; or cytosine deaminase acting on tRNA (CDAT), and (ii) the adenosine deaminase is selected from the group consisting of adenosine deaminase acting on RNA 1 (ADAR1), ADAR2, ADAR3; adenosine deaminase acting on tRNA 1 (ADAT1), ADAT2, ADAT3; and naturally occurring or engineered tRNA-specific adenosine deaminase (TadA).
  15. 15 . The method of claim 6 , wherein the targeting ribonucleic acid is a guide RNA or trigger RNA.
  16. 16 . The method of claim 11 , wherein the base editor or the targeting ribonucleic acid is encoded by one or more nucleic acid molecules administered to the subject.
  17. 17 . The method of claim 16 , wherein the targeting ribonucleic acid and/or the one or more nucleic acid molecules are administered to the subject with a lipid nanoparticle (LNP), a peptide cage, or a polymer nanoparticle.
  18. 18 . The method of claim 2 , wherein the base alteration results in a conversion of an amino acid codon to a premature stop codon to thereby down-regulate the expression of the gene.
  19. 19 . The method of claim 18 , wherein the base alteration results in the conversion of a CGA, CAG, or TGG codon to a premature TGA, TAG, or TAA stop codon, respectively, and the base editor comprises the cytidine deaminase domain.
  20. 20 . The method of claim 2 , wherein the base alteration results in a conversion of a premature stop codon to an amino acid codon to thereby up-regulate the expression of the gene.

Description

CROSS REFERENCE TO RELATED APPLICATION This application claims priority to U.S. Provisional Patent Application 63/379,512, filed Oct. 14, 2022, the disclosure of which is incorporated herein by reference. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY This application contains a sequence listing, which is submitted electronically. The content of the electronic sequence listing (065830-15WO1 Sequence Listing.xml; size: 130 KB; and date of creation: Nov. 22, 2023) is herein incorporated by reference in its entirety. FIELD OF THE INVENTION The invention relates to the field of gene therapy. In particular, it relates to controlling gene expression from an episomal vector by utilizing gene editing agents, such as base editing systems. BACKGROUND OF THE INVENTION Gene therapy in its current design is an irreversible process. It cannot be stopped in case of unwanted side effects, nor can expression levels of therapeutics be adjusted to individual patient's needs. Adeno-associated viral (AAV) vector-mediated gene therapy holds great potential for future medical applications. However, to facilitate safer and broader applicability and to enable patient-centric care, therapeutic protein expression should be controllable. For example, it has been shown in certain diseases that gene therapy in which genes become overexpressed may be toxic (Payne, Mol Ther Methods Clin Dev. 2022 March 4:25:1-2.; Palmieri et al., Front Neurosci. 2023 May 25:17:1172805). Conversely, in other diseases, such as Huntingtin's Disease, too much gene repression may be toxic (Jung et al., Hum Mol Genet. 2021 Apr. 26; 30(3-4):135-148; Wang et al., Proc Natl Acad Sci USA. 2016 Mar. 22; 113(12):3359-64; Murthy et al., PLoS Genet. 2019 March; 15(3): e1007765). In other cases, AAV gene therapy has been shown to have a current risk/benefit profile that is low. For example, in diseases with existing treatments, or diseases where patient outcomes are better or have less serious consequences (Evan et al., Curr Opin Rheumatol. 2023 Jan. 1; 35(1):37-43; Ishikawa et al., Circ Res. 2018 Aug. 17; 123(5):601-613). Thus, a method of regulating gene therapy in such diseases is needed. Base editing is a genome editing method that directly generates precise point mutations in genomic DNA or in cellular RNA without directly generating double-strand breaks (DSBs), requiring a DNA donor template or relying on cellular homology directed repair. Since base editors do not normally create DSBs, they minimize the formation of DSB-associated byproducts (Komor, A. C. et al, (2017) Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity, Sci Adv 3, and Rees, H. A. et al., (2017) Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery, Nat. Commun. 8, 15790). Base editors (BEs) are typically fusions of a Cas (“CRISPR-associated”) domain and a nucleobase modification domain. Two major types of base editors have been developed and widely used. The first type includes cytosine base editors (CBEs), which were first reported in 2016 (Komor et al., 2016; (2016) Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells. Nat. Methods, 13, 1029-1035; Nishida et al., (2016) Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science, 353, aaf8729). The second type includes adenine base editors (ABEs) that were first described in 2017 (Gaudelli et al., (2017) Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature, 551, 464-471). Both CBEs and ABEs were based on the CRISPR-Cas9 system, utilizing cytidine deaminases and adenine deaminases to confer C-to-T and A-to-G base transition changes in the editing windows respectively. CBEs can convert four codons (CGA, CAG, GAG, GAA and TGG) into stop codons (TGA, TAG, TAA) (Kuscu et al., (2017) CRISPR-STOP: gene silencing through base-editing-induced nonsense mutations. Nat. Methods, 14, 710-712; Molla and Yang, (2019) CRISPR/Cas-mediated base editing: technical considerations and practical applications. Trends Biotechnol. 37, 1121-1142). Hence, CBEs can be used for knocking out protein-coding genes by introducing premature stop codons. The present disclosure provides methods of regulating expression of a gene located on an episomal vector in a subject. In particular, the present disclosure provides a method of regulating expression of a gene located on an episomal vector utilizing a base editor system. BRIEF SUMMARY OF THE INVENTION The disclosure provides methods of regulating expression of a gene located on an episomal vector in a subject in need thereof, comprising administering to the subject one or more gene editing agents that modify a region of the gene to thereby regulate the expression of the gene, wherein the region of the gene comprises one or more of a promoter, an enhan