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WO2024152058A1 - Compositions et méthodes pour le traitement de la tyrosinémie héréditaire de type 1 - Google Patents

Compositions et méthodes pour le traitement de la tyrosinémie héréditaire de type 1 Download PDF

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WO2024152058A1
WO2024152058A1 PCT/US2024/011693 US2024011693W WO2024152058A1 WO 2024152058 A1 WO2024152058 A1 WO 2024152058A1 US 2024011693 W US2024011693 W US 2024011693W WO 2024152058 A1 WO2024152058 A1 WO 2024152058A1
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editing
hpd
grna
gene
enzyme
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PCT/US2024/011693
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Kiran Musunuru
Madelynn WHITTAKER
Xiao Wang
William PERANTEAU
Cara BERKOWITZ
Ana Maria DUMITRU
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The Trustees Of The University Of Pennsylvania
The Children's Hospital Of Philadelphia
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    • C12Y307/00—Hydrolases acting on carbon-carbon bonds (3.7)
    • C12Y307/01—Hydrolases acting on carbon-carbon bonds (3.7) in ketonic substances (3.7.1)
    • C12Y307/01002—Fumarylacetoacetase (3.7.1.2)
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    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention relates base editing therapies for the treatment of hereditary tyrosinemia type 1 (HT1). More specifically, the invention relates to methods and compositions for the inactivation of enzymes in the tyrosine (Tyr) catabolic pathway in vivo, both prenatally and postnatally, to prevent accumulation of toxic fumarylacetoacetate hydrolase (FAH) derivatives.
  • HT1 hereditary tyrosinemia type 1
  • Hereditary tyrosinemia type 1 is an autosomal recessive metabolic liver disease that can cause death in the first months of life and incurs an increased risk of hepatocellular cancer (HCC). It results from mutations in the gene encoding FAH, the last enzyme in the tyrosine (Tyr) catabolic pathway, resulting in accumulation of toxic metabolites.
  • NTBC nitisinone
  • HPD 4-hydroxyphenylpyruvate dioxygenase
  • Phe phenylalanine
  • adenoviral-associated vector comprising CRISPR-mediated base editor and a guide RNA (gRNA) or lipid nanoparticles comprising adenosine base editor (ABE) or a cytosine base editor (CBE) mRNA and at least one gRNA targeting an enzyme in the tyrosine catabolic pathway, the gRNA specifically hybridizing to a nucleic acid encoding said enzyme in the tyrosine catabolic pathway; and introducing a modified codon in the enzyme by base editing, thereby reducing expression of said enzyme in the tyrosine catabolic pathway, and alleviating symptoms of tyrosinemia type I in said subject.
  • gRNA guide RNA
  • ABE adenosine base editor
  • CBE cytosine base editor
  • the base editor comprises SpCas9 and the gRNA targets 4-hydroxyphenylpyruvate dioxygenase (HPD) and comprises at least one of the sequences recited in tables 2-4.
  • the CRISPR-mediated base editor is BE3.
  • the base editor is an ABE and catalyzes a specific A to G conversion in said nucleic acid encoding enzyme, wherein said gRNA targets HPD and comprises at least one of the sequences recited in tables 2 or 4 or Figure 3 or Figure 9C.
  • the gRNA is HPD20 of SEQ ID NO: 88.
  • the base editor is an CBE and catalyzes a specific C to T conversion in said nucleic acid encoding enzyme, wherein said gRNA targets HPD and comprises at least one of the sequences recited in table 3.
  • the editing introduces a base deletion or a base insertion in the HPD gene thereby inactivating HPD.
  • the subject is a fetus that is inside the uterus of a body of a living carrier.
  • the administration is performed postnatally in an infant, adult or pediatric.
  • the methods provided herein decrease the risk of hereditary tyrosinemia type 1 (HT1) disorder.
  • HT1 hereditary tyrosinemia type 1
  • the introduction of a modified codon in the HPD gene effects a permanent gene knockout of the HPD gene and the permanent HPD gene knockout restores normal liver function in the subject prior to birth or after birth.
  • the introduction of a modified codon in the FAH gene corrects the mutation in said FAH encoding nucleic acid and restores FAH activity which treats hereditary tyrosinemia type I (HT1) disorder in the subject prior to birth.
  • the base editing occurs in liver cells.
  • the base editing may be a modification of a C to T on a sense strand, producing a nonsense codon, or a G to A on an antisense strand, also resulting in a nonsense codon.
  • the base editor is an ABE which converts an A to a G on either the sense or anti-sense strand, thereby disrupting a splice site in said nucleic acid encoding said enzyme or the base editor is an CBE (which converts an C to a T on either the sense or anti-sense strand, thereby disrupting a splice site in said nucleic acid encoding said enzyme.
  • the ABE which converts an G to an A in the FAH encoding nucleic acid restores FAH enzyme activity.
  • the editing is performed via delivery of an adenoviral vector selected from AAV8 and AAV9 or via delivery of a lipid nanoparticle.
  • kits for practicing any of the methods described herein can comprise at least one AAV vector or lipid nanoparticle carrying at least one of the base editors and targeting gRNAs described above. Suitable buffers and negative control gRNAs can also be included.
  • BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: Tyrosine catabolic pathway.
  • Figures 2A-2F In utero base editing rescues HT1 mouse model. An adenoviral vector containing a cytosine base editor (BE3) to introduce a nonsense mutation in the HPD gene was injected into gestational day (E) 16 HT1 fetuses11.
  • FIG. 2A Next-generation sequencing to assess % edited alleles in liver.
  • FIG. 2A Next-generation sequencing to assess % edited alleles in liver.
  • FIG. 2B-2D Liver function, weight gain, and survival of HT1 mice on NTBC compared to prenatally edited and unedited HT1 mice off NTBC.
  • FIG. 2E, 2F Liver immunohistochemistry for and quantification of HPD protein expression in prenatally edited HT1 mice and wild-type Balb/c mice at 3 months of age. Scale bar (FIG. 2E), 1 mm (left); 100 ⁇ m (right).
  • Figure 3 In vitro correction of the human FAH mutation. FAH sequence around the site of the Quebec founder G ⁇ A mutation, which is indicated with underlines.
  • BE3 was used to introduce the Quebec mutation (and an additional G ⁇ A edit, both shown in bold) in HEK 293 cells; the guide RNA protospacer is shown with a box, the PAM in pink, and the cytosine editing window in blue.
  • ABE7.1 was used to correct the Quebec mutation (A ⁇ G); the guide RNA protospacer is shown with a box, the PAM in pink, and the adenine editing window in blue. The most common edited alleles with correction are shown, along with their frequencies; a total of 9% of the 293.BE3 alleles had a corrected Quebec mutation.
  • FIGS. 4A-4C Targeting the HPD gene with SpCas9 and gRNA results in efficient editing and reduced HPD protein expression.
  • AAV8 or lipid nanoparticles (LNP) containing the SpCas9 transgene or mRNA respectively and the HPD targeting gRNA were delivered to E16 HT1 fetuses and injected mice were assessed for hepatocyte editing efficiencies and HPD protein expression.
  • FIG. 4A Scheme of experiment.
  • FIG. 4B Next-generation sequencing to assess % edited alleles in liver; blue square, LNP.SpCas9.Hpd injected, black triangle, AAV8.SpCas9.Hpd injected.
  • FIG. 4C Quantification of liver HPD protein expression via immunohistochemistry in prenatally edited HT1 mice and wild-type Balb/c mice at 4.5 months of age.
  • Figures 5A-5D Targeting the HPD gene with SpCas9 and gRNA rescues the HT1 phenotype.
  • AAV8 or LNP containing the SpCas9 transgene or mRNA respectively and the HPD targeting gRNA were delivered to E16 HT1 mouse fetuses as shown in schematic in Fig. 4A. (FIG.
  • FIG. 5A, 5B Liver function (serum alanine transaminase (ALT) and aspartate aminotransferase(AST)) was assessed in recipients of AAV.SpCas9.Hpd (FIG. 5A) and LNP.SpCas9.Hpd (FIG. 5B) at 4 months of age and compared to un-injected HT1 mice off NTBC and HT1 mice maintained on NTBC.
  • FIG. 5C, 5D Weight gain and survival of HT1 mice on NTBC compared to prenatally edited and unedited HT1 mice off NTBC.
  • Figure 6 CBE of the human HPD gene.
  • FIG. 7A-7D Dose response curve of on-target HPD editing in HuH-7 cells.
  • HuH-7 cells were transfected with ionizable lipid nanoparticles (LNP) carrying ABE8.8m mRNA and one of four sgRNAs -guides (sgRNA HPD3; FIG.
  • HuH-7 cells were treated with ABE8.8 mRNA and either HPD3 gRNA, HPD4 gRNA, HPD5 gRNA, or HPD20 gRNA described in Figure 7.
  • FIG. 8A Sanger sequencing of edited and unedited control cells demonstrates A>G editing (sense strand) or T>C editing (antisense strand) with the edited base highlighted in the blue box.
  • FIG. 8B In a separate experiment, on target editing in HuH-7 cells was assessed following transfection with ABE8.8 mRNA and HPD3, HPD4, HPD5, or HPD20 gRNA.
  • FIG. 8C Protein was isolated from cells in the experiments in Fig 2b and assessed by Western blot for HPD protein levels.
  • FIGS 9A-9D Off-target assessment of ABE8.8/HPD3 and ABE8.8/HPD20. Targeted amplicon sequencing of the top ONE-seq nominated sites (ONE-seq scores down to 0.01) for gRNAs HPD3 (FIG. 9A) and HPD20 (FIG. 9B) in HuH-7 cells. Systematic One-seq analyses of hybrid guide RNAs for HPD20. SEQ ID NOS: 88 - 109 are shown in descending order (FIG.
  • mice were intravascularly injected with AAV8 carrying the ABE8.8m transgene and either the muHPD345 gRNA (orthologous to human-specific guides HPD3, HPD4, and HPD5) or muHPD20 gRNA (orthologous to human-specific guide HPD20).
  • NTBC was removed from mice 2 weeks following injection. For adult recipients, NTBC was cycled back on 1 week following initial removal and then removed 1 week later without subsequent cycling. For neonatal recipients, NTBC was never resumed after initial removal at 2 weeks of life. Individual mice were weighed daily (FIG. 10A, 10B) and survival following NTBC removal was noted (FIG. 10C, 10D).
  • Control mice consisted of HT1 mice that were not edited and from which NTBC was removed according to the same schedule of the age-matched experimental mice.
  • Figure 11A-11C In vivo editing and improved liver function in HT1 neonatal mice following AAV8.ABE8.8.muHPD345 injection. Day of life 1 HT1 mice were intravascularly injected with AAV8 carrying the ABE8.8 transgene and the muHPD345 gRNA. NTBC was removed from mice 2 weeks following injection.
  • FIG. 11A Injected mice were sacrificed 2 months after NTBC removal and the DNA from the liver and other organs was assessed for on- target A>G editing via next generation sequencing.
  • FIG. 12A Efficient in vivo editing and phenotypic rescue with LNP.muHPD20.
  • NTBC was withdrawn at 2 weeks of age.
  • FIG. 12A Sanger and next-generation sequencing to assess edited HPD alleles in the liver 70 days after NTBC removal.
  • FIG. 12B Editing of HPD alleles in other organs at 70 days off NTBC.
  • FIG. 12C-12E Liver function tests including total bilirubin (FIG. 12C), AST (FIG. 12D), and ALT (FIG. 12E) were assessed at necropsy in edited HT1 mice off NTBC and unedited control HT1 mice on and off NTBC.
  • Figures 13A-13H LNP targeting of the fetal liver.
  • FIG. 13A-13H LNPs with GFP mRNA were intravenously injected into E16 fetal mice (FIG. 13B), 0.5 gestation fetal NHPs (FIG. 13D, 13F) and E70 fetal sheep (FIG. 13H). Livers were harvested ⁇ 24 hours later and assessed for GFP by immunohistochemistry. The controls (FIGs. 13A, 13C, 13E, 13G) were un- injected. Scale bar, 1 mm (FIG. 13C, 13D, 13G, 13H); 100 ⁇ m (FIG. 13E, 13F).
  • Figures 14A-14E Fetal Hpd inactivation via LNP delivery of SpCas9 rescues the HT1 mouse model.
  • FIG. 14A NGS to assess edited Hpd alleles.
  • FIG. 14B, 14C Liver function and survival of HT1 mice on NTBC compared to prenatally edited and unedited HT1 mice off NTBC (FIG. 14D, 14E). Immunohistochemistry for and quantification of liver HPD protein expression in prenatally edited HT1 mice and control Balb/c mice at 5 months of age. Scale bar, 1mm.
  • FIGS. 15A – 15C LNP delivery of ABE8.8 mRNA HPD20 gRNA in adult HT1 mice results in efficient hepatocyte editing.
  • ABE8.8 mRNA and mouse orthologous HPD20 gRNA were packaged in an LNP for intravascular delivery and in vivo base editing.
  • FIG. 15A The experimental scheme.
  • FIG. 15B Baseline hepatocyte editing was assessed in Balb/c mice in which no survival advantage exists for edited cells. Two weeks post injection, DNA from the liver was assessed by next generation sequencing for on-target A>G editing and noted approximately 60% baseline editing.
  • FIG. 15A The experimental scheme.
  • FIG. 15B Baseline hepatocyte editing was assessed in Balb/c mice in which no survival advantage exists for edited cells. Two weeks post injection, DNA from the liver was assessed by next generation sequencing for on-target A>G editing and noted approximately 60% baseline editing.
  • FIG. 16B HT1 mice were edited using LNPs containing ABE8.8 mRNA and HPD20 gRNA at either 1 mg/kg or 2 mg/kg. Urine was analyzed at 2 months post NTBC removal and assessed for urine metabolites. HT1 edited mice demonstrated an increase in urine HPP (FIG 16B) and reduction in urine SA (FIG: 16C) consistent with editing efficiently inactivating the HPD gene.
  • FIGS 17A – 17D In vivo base editing via LNP delivery of ABE8.8 mRNA and HPD20 gRNA in adult HT1 mice improves liver function, weight gain and rescues the lethality associated with HT1. 8 week old HT1 mice were injected with LNPs containing ABE8.8 mRNA and HPD20 gRNA at either 1 mg/kg or 2 mg/kg. Serum was assessed at 2 months following NTBC removal for AST (Fig 11A) and ALT (Fig 11B). Edited mice demonstrated normalization of liver function similar to unedited HT1 mice maintained on NTBC and in contrast to unedited HT1 mice off NTBC in which there was a significant elevation in ALT and AST.
  • FIG. 18A In utero baseline adenine editing via LNP or AAV8 delivery and the orthologous HPD20 sgRNA in the liver.
  • FIG. 18B A comparison of fetal baseline liver editing and (FIG. 18B) percentage on target editing following AAV delivery.
  • AAV8 delivery in fetal HT1 mice also rescued the phenotype (FIG. 18C) fetal AST levels and (FIG. 18D) fetal ALT levels. Treated mice also exhibited improved survival and weight gain.
  • FIGS. 19A - 19 Fetal liver function improves and maternal liver function is normal as determined by AST and ALT levels after in utero base editing for HT1 in a non- human primate model.
  • FIG. 19A Schematic diagram of treatment protocol.
  • FIG. 19B On target base editing in the liver and
  • FIG. 19C reduced liver HPD protein levels.
  • FIGS. 19D and 19E Liver function measurements in maternal (FIGS. 19D and 19E) and fetal samples (FIGS. 19F and 19G)
  • Figures 20A- 20B In utero base editing for HT1 in postnatal nonhuman primate.
  • NHPs Macaca fascicularis nonhuman primates
  • LNPs lipid nanoparticles
  • AAV adeno-associated virus
  • SpCas9 Streptococcus pyogenes Cas9
  • SpCas9 transgene Streptococcus pyogenes Cas9
  • gRNA mouse HPD gene targeting gRNA
  • LNP or AAV mediated delivery of adenine base editor 6.3 (ABE6.3) mRNA/transgene and a gRNA targeting the G ⁇ A mutation in the murine FAH gene results in correction of the underlying mutation in the mouse liver and rescue of the HT1 phenotype.
  • splice sites in the human HPD gene amenable to high- efficiency editing with cytosine base editor (BE4) or adenine base editor 8.8-m (ABE8.8), with several gRNA candidates achieving substantial reductions in HPD protein levels in a human hepatocyte cell line.
  • AAV or LNP delivery of ABE8.8 mRNA and mouse gRNAs orthologous to the human gRNAs capable of disrupting human HPD gene splice sites demonstrated substantial whole-liver HPD editing in vivo.
  • the terms “component,” “composition,” “composition of compounds,” “compound,” “drug,” “pharmacologically active agent,” “active agent,” “therapeutic,” “therapy,” “treatment,” or “medicament” are used interchangeably herein to refer to a compound or compounds or composition of matter which, when administered to a subject (human or animal) induces a desired pharmacological and/or physiologic effect by local and/or systemic action.
  • treatment or “therapy” (as well as different forms thereof) include preventative (e.g., prophylactic), curative or palliative treatment.
  • the term “treating” includes alleviating or reducing at least one adverse or negative effect or symptom of a condition, disease or disorder.
  • subject means a human, to whom treatment, including prophylactic treatment, with the pharmaceutical composition according to the present invention, is provided.
  • subject refers to human and non-human animals.
  • non-human animals and “non-human mammals” are used interchangeably herein and include all vertebrates, e.g., mammals, such as non-human primates, (particularly higher primates), sheep, dog, rodent, (e.g.
  • polynucleotide refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • a polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • nucleotide structure may be imparted before or after assembly of the polymer.
  • sequence of nucleotides may be interrupted by non nucleotide components.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • wild type is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.
  • variant should be taken to mean the exhibition of qualities that have a pattern that deviates from the wild type or a comprises non naturally occurring components.
  • nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.
  • “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types.
  • a percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).
  • Perfectly complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%.
  • an effective amount refers to the amount of an agent that is sufficient to effect beneficial or desired results.
  • the therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • the term also applies to a dose that will provide an image for detection by any one of the imaging methods described herein.
  • the specific dose may vary depending on one or more of: the particular agent chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to be imaged, and the physical delivery system in which it is carried.
  • the practice of the present invention employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al.
  • PCR 2 A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).
  • Vectors can be designed for expression of CRISPR transcripts (e.g.
  • CRISPR transcripts can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press. San Diego, Calif. (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • a vector is capable of driving expression of one or more sequences in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195).
  • the expression vector's control functions are typically provided by one or more regulatory elements.
  • commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art.
  • tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl.
  • albumin promoter liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277
  • lymphoid-specific promoters Calame and Eaton, 1988. Adv. Immunol. 43: 235-275
  • pancreas-specific promoters Eslund, et al., 1985. Science 230: 912-916
  • mammary gland-specific promoters e.g., milk whey promoter, U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166
  • Developmentally regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).
  • CRISPR system refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a "direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a "spacer” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus.
  • a tracr trans-activating CRISPR
  • tracr-mate sequence encompassing a "direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system
  • guide sequence also referred to as a "spacer” in the context of an endogenous CRISPR system
  • one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system).
  • target sequence refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex.
  • a target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides.
  • a target sequence is located in the nucleus or cytoplasm of a cell.
  • the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or chloroplast.
  • a sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an "editing template” or “editing polynucleotide” or “editing sequence”.
  • an exogenous template polynucleotide may be referred to as an editing template.
  • the recombination is homologous recombination.
  • one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites.
  • a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors.
  • two or more of the elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector.
  • CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5' with respect to ("upstream" of) or 3' with respect to ("downstream" of) a second element.
  • the coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction.
  • a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g. each in a different intron, two or more in at least one intron, or all in a single intron).
  • the CRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter.
  • a vector comprises one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a "cloning site").
  • one or more insertion sites are located upstream and/or downstream of one or more sequence elements of one or more vectors.
  • a vector comprises an insertion site upstream of a tracr mate sequence, and optionally downstream of a regulatory element operably linked to the tracr mate sequence, such that following insertion of a guide sequence into the insertion site and upon expression the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell.
  • a vector comprises two or more insertion sites, each insertion site being located between two tracr mate sequences so as to allow insertion of a guide sequence at each site.
  • the two or more guide sequences may comprise two or more copies of a single guide sequence, two or more different guide sequences, or combinations of these.
  • a single expression construct may be used to target CRISPR activity to multiple different, corresponding target sequences within a cell.
  • a single vector may comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guide sequences.
  • a vector comprises a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein.
  • Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2.
  • These enzymes are known; for example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2.
  • the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9.
  • the CRISPR enzyme is Cas9, and may be Cas9 from S. pyogenes or S. pneumoniae.
  • the CRISPR enzyme directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.
  • a vector encodes a CRISPR enzyme that is mutated to with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence.
  • an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand).
  • nickases may be used for genome editing via homologous recombination.
  • an enzyme coding sequence encoding a CRISPR enzyme is codon optimized for expression in particular cells, such as eukaryotic cells.
  • the eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate.
  • codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence.
  • codon bias differs in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RNA
  • Codon usage tables are readily available, for example, at the "Codon Usage Database", and these tables can be adapted in a number of ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000).
  • Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available.
  • one or more codons in a sequence encoding a CRISPR enzyme correspond to the most frequently used codon for a particular amino acid.
  • a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence.
  • the degree of complementarity between a guide 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.
  • a tracr mate sequence includes any sequence that has sufficient complementarity with a tracr sequence to promote one or more of: (1) excision of a guide sequence flanked by tracr mate sequences in a cell containing the corresponding tracr sequence; and (2) formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises the tracr mate sequence hybridized to the tracr sequence.
  • the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR enzyme).
  • a CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains.
  • epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
  • reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP).
  • a CRISPR enzyme may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US20110059502, incorporated herein by reference. In some embodiments, a tagged CRISPR enzyme is used to identify the location of a target sequence.
  • MBP maltose binding protein
  • DBD Lex A DNA binding domain
  • HSV herpes simplex virus
  • a reporter gene which includes but is not limited to glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and auto-fluorescent proteins including blue fluorescent protein (BFP), may be introduced into a cell to encode a gene product which serves as a marker by which to measure the alteration or modification of expression of the gene product.
  • GST glutathione-5-transferase
  • HRP horseradish peroxidase
  • CAT chloramphenicol acetyltransferase
  • beta-galactosidase beta-galactosidase
  • beta-glucuronidase beta-galactosidase
  • the DNA molecule encoding the gene product may be introduced into the cell via a vector.
  • the gene product is luciferase.
  • the expression of the gene product is decreased.
  • the invention provides methods comprising delivering one or more polynucleotides, such as or one or more vectors as described herein, one or more transcripts thereof, and/or one or proteins transcribed therefrom, to a host cell.
  • the invention further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells.
  • a CRISPR enzyme in combination with (and optionally complexed with) a guide sequence is delivered to a cell.
  • Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of a CRISPR system to cells in culture, or in a host organism.
  • Non-viral vector delivery systems include DNA plasmids, RNA (e.g. a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome.
  • Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
  • Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.
  • Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam TM and Lipofectin TM ).
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).
  • lipid:nucleic acid complexes including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.
  • GalNAc-Lipid nanoparticles which enable non-LDLR dependent hepatic delivery of a CRISPR base editing therapy in Nat Commun 14, 2776 (2023).
  • suitable lipid nanoparticle formulations see also US Patent No. 11,801,306 and International Patent Application No. WO2022060871.
  • Clinical trials are also being conducted which also employ a LNP described in trial clinical trial identifier no. NCT05398029.
  • Virus-like Particles as Nanocarriers for Intracellular Delivery of Biomolecules and Compounds are described by He, J. et al. in “Virus-like Particles as Nanocarriers for Intracellular Delivery of Biomolecules and Compounds.” Viruses. 2022 Aug 28;14(9):1905.
  • RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus.
  • Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo).
  • Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene.
  • Retroviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression.
  • Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immune-deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol.
  • MiLV murine leukemia virus
  • GaLV gibbon ape leukemia virus
  • SIV Simian Immuno deficiency virus
  • HAV human immune-deficiency virus
  • adenoviral based systems may be used.
  • Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system.
  • Adeno-associated virus vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No.
  • Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and ⁇ 2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle.
  • the vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed.
  • the missing viral functions are typically supplied in trans by the packaging cell line.
  • AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome.
  • Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
  • the cell line may also be infected with adenovirus as a helper.
  • the helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid.
  • the helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.
  • a host cell is transiently or non-transiently transfected with one or more vectors described herein.
  • a cell is transfected as it naturally occurs in a subject.
  • a cell that is transfected is taken from a subject.
  • the cell is derived from cells taken from a subject, such as a cell line.
  • the invention provides for methods of modifying a target polynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or in vitro.
  • the method comprises sampling a cell or population of cells from a human or non-human animal, and modifying the cell or cells. Culturing may occur at any stage ex vivo. The cell or cells may be re- introduced into the human or non-human animal.
  • the invention provides for methods of modifying a target polynucleotide in a eukaryotic cell.
  • the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence.
  • the invention provides kits containing any one or more of the elements disclosed in the above methods and compositions.
  • the kit comprises a vector system or components for an alternative delivery system such as those described above and instructions for using the kit.
  • the vector or delivery system comprises (a) a first regulatory element operably linked to a tracr mate sequence and one or more insertion sites for inserting a guide sequence upstream of the tracr mate sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence; and/or (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence.
  • kits may be provided individually or in combinations, and may be provided in any suitable container, such as a vial, a bottle, or a tube.
  • the kit includes instructions in one or more languages, for example in more than one language.
  • a kit comprises one or more reagents for use in a process utilizing one or more of the elements described herein.
  • Reagents may be provided in any suitable container.
  • a kit may provide one or more reaction or storage buffers.
  • Reagents may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g. in concentrate or lyophilized form).
  • a buffer can be any buffer, including but not limited to a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof.
  • the buffer is alkaline.
  • the buffer has a pH from about 7 to about 10.
  • the kit comprises one or more oligonucleotides corresponding to a guide sequence for insertion into a vector so as to operably link the guide sequence and a regulatory element.
  • the kit comprises a homologous recombination template polynucleotide.
  • the invention provides methods for using one or more elements of a CRISPR system.
  • the CRISPR complex of the invention provides an effective means for modifying a target polynucleotide.
  • the CRISPR complex of the invention has a wide variety of utility including modifying (e.g., deleting, inserting, translocating, inactivating, activating) a target polynucleotide in a multiplicity of cell types in methods of gene therapy.
  • An exemplary CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within the target polynucleotide.
  • the guide sequence is linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence.
  • Hereditary Tyrosinemia type I is a fatal genetic disease caused by autosomal recessive mutations in the Fah gene, which codes for the fumarylacetoacetate hydroxylase (FAH), leading to the accumulation of toxic fumarylacetoacetate and succinyl acetoacetate, causing liver and kidney damage.
  • FH fumarylacetoacetate hydroxylase
  • NTBC 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione
  • NTBC 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione
  • this treatment requires lifelong management of diet and medication and may eventually require liver transplantation.
  • the invention provides a method for in vivo genome editing, both prenatal and postnatal, the method comprising: administering to a subject an adenoviral vector, wherein the subject is a fetus, the adenoviral vector comprising CRISPR-mediated base editor 3 (BE3) and a guide RNA (gRNA), the gRNA targeting an enzyme in the tyrosine catabolic pathway; and introducing a modified codon in the therapeutic gene by base editing the therapeutic gene, wherein the base editing is performed by the adenoviral vector or lipid nanoparticle.
  • Base editing addresses the disadvantage of the need to create double strand breaks (DSBs) to instigate NHEJ or HDR.
  • the base editor can comprise a catalytically impaired Streptococcus pyogenes Cas9 (SpCas9) protein, unable to make DSBs, fused to either a cytosine deaminase domain from a nucleic acid-editing protein (CBE) or a modified tRNA adenosine deaminase (ABE).
  • SpCas9 and gRNA tether the base editor at the genome target site, and the cytosine deaminase converts a nearby cytosine into uracil and, ultimately, thymine (resulting in either C ⁇ T or G ⁇ A changes in the coding sequence of a gene, depending on which strand is targeted).
  • the cytosine deaminase can introduce nonsense mutations in a site-specific fashion.
  • the adenine deaminase converts a nearby adenine into inosine and, ultimately, guanine and can correct a disease-causing G ⁇ A mutation.
  • base editing does not require proliferating cells to efficiently introduce mutations.
  • the method further comprises before step (a) identifying in vitro a target codon for base editing into a nonsense codon; and generating the adenoviral vector by cloning BE3-encoding gene, a synthetic polyadenylation sequence from pCMV-BE3, CAG reporter from pCas9_GFP, and U6 promoter-driven gRNA cassette with a protospacer sequence into a dual- expression vector.
  • the gRNA targets the splice acceptor or the splice donor.
  • the gRNA is at least one of those identified in tables 2- 4.
  • genome editing is performed in utero when the fetus is inside a uterus of its carrier and the uterus is inside the body of a living carrier.
  • the carrier may be a mammal.
  • the mammal may be an animal.
  • the mammal may be human.
  • the living carrier may be the mother of the fetus or a surrogate.
  • genome editing is performed in utero when the fetus is inside a uterus, and the uterus is outside the body of a carrier, e.g., not within the body of any living carrier, for example the uterus is in vitro or in an alternate embodiment, the uterus is ex vivo.
  • the target codon is screened for a glutamine residue and a tryptophan residue, wherein the glutamine and tryptophan residues are within a base editing window of a protospacer adjacent motif (PAM) of the BE3, wherein the window is flanked by four proximal and four distal bases, wherein the proximal and distal bases match reference sequences.
  • the CRISPR-mediated base editor is base editor 4 (BE4) rather than BE3.
  • the method further comprises assessing C bases within the window for a change to another base.
  • the gRNA is selected if the BE3 PAM sequence (NGG) is 13-17 nucleotides distal to the target cytosine base(s).
  • the change is via a C to T on a sense strand, and the modified codon is changed to a nonsense codon.
  • the change is via a G to A on an antisense strand, and the modified codon is changed to a nonsense codon.
  • the change is via a C to T on a sense strand, and the change is a missense variant.
  • the change is via a G to A on an antisense strand, and the change is a missense variant.
  • the base editing may occur prior to disease onset, wherein the disease is a phenotype resulting from a mutation in the therapeutic gene. In certain embodiments, the base editing decreases a risk of developing a disease.
  • the therapeutic gene is a 4-hydroxyphenylpyruvate dioxygenase (HPD) gene.
  • the gRNAs are selected from the sequences identified in Tables 2-4.
  • the introduction of a modified codon (e.g., a nonsense codon) in the HPD gene effects a permanent gene knockout of the Hpd gene.
  • the permanent HPD gene knockout restores normal liver function in the subject prior to birth.
  • the permanent HPD gene knockout treats hereditary tyrosinemia type I (HT1) disorder in the subject prior to birth.
  • HT1 hereditary tyrosinemia type I
  • the invention also provides a method for treating a HT1 in a subject, the method comprising: identifying in vitro a target codon for base editing; generating the adenoviral vector by cloning BE3-encoding gene, a synthetic polyadenylation sequence from pCMV-BE3, CAG reporter from pCas9_GFP, and U6 promoter-driven gRNA cassette with a protospacer sequence into a dual-expression vector; administering to the fetal subject an adenoviral vector, the adenoviral vector comprising CRISPR-mediated base editor 3 (BE3) and a guide RNA (gRNA), and the gRNA targeting an enzyme in the tyrosine catabolic pathway; and introducing a modified codon in the therapeutic gene by base editing the therapeutic gene, wherein the base editing is performed by the adenoviral vector.
  • a target codon for base editing comprising: identifying in vitro a target codon for base editing; generating the adenoviral vector by
  • the enzyme is HPD.
  • the CRISPR-mediated base editor is base editor 4 (BE4) instead of BE3.
  • the target codon is screened for a glutamine residue and a tryptophan residue, wherein the glutamine and tryptophan residues are within a base editing window of a protospacer adjacent motif (PAM) of the BE3, wherein the window is flanked by four proximal and four distal bases, wherein the proximal and distal bases match reference sequences.
  • the method further comprises assessing C bases within the window for a change to another base.
  • the gRNA is selected if the BE3 PAM sequence (NGG) is 13-17 nucleotides distal to the target cytosine base(s).
  • the change is via a C to T on a sense strand, and the modified codon is changed to a nonsense codon.
  • the change is via a G to A on an antisense strand, and the modified codon is changed to a nonsense codon.
  • the change is via a C to T on a sense strand, and the change is a missense variant.
  • the change is via a G to A on an antisense strand, and the change is a missense variant.
  • the base editing occurs prior to disease onset, wherein the disease is a phenotype resulting from the mutation in the therapeutic gene.
  • the base editing may decrease a risk of developing HT1.
  • Any patent, patent application publication, or scientific publication, cited herein, is incorporated by reference herein in its entirety. The examples are presented in order to more fully illustrate embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. Materials and Methods Animals Balb/c, C57Bl/6 (called B6) and Fah 1R Tyr c /RJ (called FAH; stock #018129) mice were purchased from Jackson Laboratories (Bar Harbor, ME).
  • FAH mice were provided as heterozygotes and subsequently bred to homozygosity (Fah –/– ) in our animal facility and maintained on nitisinone (NTBC, CAS# 104206-65-7, Yecuris) in their drinking water at a concentration of 16.5 mg/L.
  • NTBC nitisinone
  • Genotyping FAH mice were genotyped to confirm the Fah –/– genotype. At weaning or the time of sacrifice, 2 mm tail snips were placed in 100 ⁇ L of 1x Lysis buffer (50x Lysis buffer: 1.25M NaOH, 10 mM EDTA) and incubated at 95°C for 1 hour. 100 ⁇ L of Neutralization buffer (50x Neutralization buffer: 2M tris-HCl) was then added and samples were vortexed.
  • 1x Lysis buffer 50x Lysis buffer: 1.25M NaOH, 10 mM EDTA
  • Neutralization buffer 50x Neutralization buffer: 2M tris-HCl
  • Extracted DNA was amplified using primers Fah-F (TCTCCCCCGCACTTAGTTTCC; SEQ ID NO: 1) and Fah- R (GGACTCAGATGCTGGGCTGATG; SEQ ID NO: 2) and PCR products were digested with the BsrBI restriction enzyme (#R0102, New England BioLabs) according to the manufacturer’s instructions. Digested samples were run on ethidium-bromide stained 1.2% agarose gels for analysis. The mutated Fah allele loses the BsrBI cut site. The genotype was also confirmed with Sanger sequencing.
  • the LNPs were serially diluted from a stock to the respective concentrations in 60 ⁇ L/well of Optimem (serum-free media) and these dilutions were added to individual wells containing 440 ⁇ L of F12 media, with total concentrations ranging on initial screen from 1 ⁇ g/mL down to subnanomolar/mL on subsequent screens. Plates were incubated for 72hrs at 370C prior to collection. For cell harvests, each well was washed with 500 ⁇ L PBS; subsequently, 500 ⁇ L of 0.25% trypsin was added to each well and the cells were incubated for 5min at 370C.
  • the forward primers used for the human guides 3,4, and 5 are as follows: 5’ ACACACCCTGGTGGAGAAGAT 3’ for qPCR (SEQ ID NO: 7) 5’TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACACACCCTGGTGGAGA AGAT 3’ for NGS (SEQ ID NO: 29)
  • the reverse primers used for the human guides 3,4, and 5 are as follows: 5’ GGGGCATTACTTTTCACAGTCC 3’ for qPCR (SEQ ID NO: 8) 5’ TGCTGTGAAAAGTAATGCC 3’ for qPCR (SEQ ID NO: 30) 5’ ACTTTTCACAGCACCTCAGGG 3’ for qPCR (SEQ ID NO: 31) 5’GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGGGCATTACTTTTCAC AGTCC 3’ for NGS (SEQ ID NO: 32)
  • the forward primers used for the human guide 20 are as follows: 5’ GCCCTCACCACTTCCCCAAT 3’
  • Genome editing to inactivate the HPD gene provides a durable, mutation-agnostic treatment for all HT1 patients.
  • the primary therapeutic described herein inactivates the HPD gene via adenine base editing.
  • Patients with acute HT1 can present within weeks of life, and NTBC with dietary therapy is the current standard of care. Despite early treatment, some patients remain at increased risk for liver failure and HCC 15,17-21 .
  • Cirrhosis has emerged in patients started on NTBC prior to 6 months of age 15 . Additionally, an infant was reported to develop HCC by 6 months of age despite starting NTBC at 2 weeks of age 18 . These cases argue for a possible benefit of earlier treatment of HT1, namely before birth.
  • the developing fetus has properties that make it ideal for in utero genome editing (IUGE): small fetal size allows delivery of a high dose of the therapeutic per body weight; progenitor cells of multiple organs, including liver, are abundant and accessible 15-17 ; and the fetal immune system is tolerant. Multiple studies demonstrate the lack of an immune response to viral vectors and expressed transgenes, including Cas9, following IUGE 11,18-20 .
  • IUGE can treat HT1 prior to the onset of pathology.
  • Pharmacologic, toxicologic, and off-target assessment approaches are used to determine the safety of the gRNA administration.
  • the data presented herein indicates that a one-time intravenous infusion of LNPs carrying ABE8.8 mRNA and a gRNA targeting a splice site in the HPD gene will sufficiently inactivate the HPD gene in hepatocytes to result in durable, if not lifelong, amelioration of HT1 disease.
  • the therapeutic consequences include the normalization of liver and renal function, reduced risk of HCC, and the ability of patients to cease NTBC.
  • the data indicates that in vivo editing better mitigates HT1 disease phenotypes without significant toxicity to the patient or, when performed in utero, to the mother or fetus.
  • the ideal clinical population for the HPD inactivating therapeutic would be all patients with HT1, independent of the underlying FAH mutation. This population includes adults on NTBC and a Tyr- and Phe-restricted diet, and children diagnosed with HT1. Although the therapy would be expected to halt progression of disease and thus benefit patients late in the disease course, the ideal therapeutic window would be early in the disease process, prior to any liver or renal damage. In addition to children as candidates for treatment, HT1 fetuses could be candidates.
  • HT1 is part of the newborn screening routinely performed in all 50 states in the U.S.
  • the finding of elevated confirmatory urine and/or serum SA levels is diagnostic, and FAH genetic testing is frequently performed for confirmation.
  • HT1 can be prenatally diagnosed via biochemical (amniotic fluid SA levels) and genetic assays (FAH mutation analysis). Additionally, families with a previous HT1-diagnosed sibling often seek out prenatal diagnosis 2,9,12-14 .
  • the prevalence of HT1 is ⁇ 1:100,000, with certain regions having a higher prevalence (Quebec, Canada, 1:16,000; Saguenay-Lac Saint-Jean region of Quebec, 1:1850) 9 .
  • mice maintained on NTBC were mated to produce time-dated pregnancies.
  • Gestational day 16 (E16) fetuses were intravascularly injected via the vitelline vein with either an LNP containing SpCas9 mRNA and a gRNA targeting the murine HPD gene or an AAV8 vector containing the SpCas9 transgene and the same gRNA.
  • E16 epistational day 16
  • injected mice were fostered with dams that were not being maintained on NTBC.
  • Mice were sacrificed at either 2 weeks of age or 4 months of life and assessed by next-generation sequencing for on- target HPD editing in DNA obtained from hepatocytes.
  • mice Prenatally edited HT1 mice, unedited HT1 mice, and wild-type Balb/c mice were monitored for weight gain, survival, and liver function following NTBC removal at 2 weeks of age. There was significant improvement in the phenotype of prenatally edited HT1 mice independent of an AAV or LNP delivery approach (Figure 5). Specifically, all unedited HT1 mice died by 51 days of life following removal of NTBC at 2 weeks of age. In contrast, the edited mice had survival was similar to HT1 mice maintained on NTBC. Similarly, weight gain of prenatally edited HT1 mice was similar to HT1 mice maintained on NTBC.
  • liver function as measured by serum ALT, AST, and bilirubin levels of prenatally edited HT1 mice off NTBC at 4.5 months of age was 1) normal, 2) similar to age- matched HT1 mice maintained on NTBC, and 3) significantly improved compared to HT1 mice off of NTBC.
  • a limited off-target analysis of the top off-target sites as predicted by sequence homology on CRISPR failed to demonstrate any significant off-target editing above background independent of the delivery vehicle. Subsequently, we demonstrated the ability to introduce a nonsense mutation in the HPD gene in hepatocytes with a CBE delivered in utero via an adenoviral vector to fetal HT1 mice ( Figure 2).
  • the initial mean on-target editing proportion was 14%, rising to 40% at 3 months following withdrawal of NTBC at birth (highlighting the survival advantage of corrected cells).
  • Prenatally edited mice off NTBC had liver function and survival at 3 months equivalent to unedited HT1 mice maintained on NTBC, while edited mice had better weight gain than unedited HT1 mice on NTBC, consistent with better overall health.
  • the edited mice had 96% reduction of HPD protein on liver immunohistochemistry.
  • cytosine and adenine base editors can be used to disrupt splice donor and acceptor sites as a mechanism of inactivating the HPD gene.
  • Table 2 gRNAs targeting disruption of HPD splice donor or acceptor sites via adenine base editing ABE8.8 editing of human HPD gene ty
  • Table 3 gRNAs targeting disruption of splice donor or acceptor sites or introduction of a nonsense mutation in the HPD gene via cytosine base editing.
  • Figure 9C shows insertion sites of 2′- deoxynucleotide (dnt) and Figure 9D shows positioning affects guide activity and specificity in a target-dependent manner and that this can be used to engineer chRDNA guides with substantially reduced off-target effects.
  • off-target analyses using ONE-seq demonstrated only one relevant site for HPD3 and HPD20 ( Figure 9A).
  • EXAMPLE 4 In vivo AAV8 delivery of ABE8.8 and mouse gRNAs orthologous to the human HPD20 gRNA and HPD 3,4,5 gRNA results in substantial on-target editing and rescue of the disease phenotype in the mouse model of HT1.
  • the murine HPD gene was assessed and gRNAs orthologous to the human HPD targeting gRNAs – HPD3, HPD4, HPD5, and HPD20 – were designed. Since HPD3, HPD4, and HPD5 all target the same splice donor site, two orthologous mouse gRNAs were designed. muHPD20 is orthologous to HPD20 and muHPD345 is orthologous to HPD3, HPD4, and HPD5.
  • the ABE8.8 transgene and either muHPD345 or muHPD20 were packaged in an AAV8 under a liver specific TTR promoter and intravascularly delivered to either neonatal or adult HT1 mice.
  • a dual AAV delivery approach with intein mediated fusion was used, similar to an approach we have used previously 12 .
  • These studies demonstrated rescue of the HT1 phenotype following in vivo editing using both the muHPD345 gRNA and muHPD20 gRNA ( Figure 10. At the moment, neonatal recipients of AAV8 containing the ABE8.8 transgene and the muHPD345 gRNA have reached their terminal end-point.
  • HT1 is a devasting genetic disease for which the only treatment is daily NTBC, a repurposed herbicide, dosing. Approximately 10% of patients don’t respond to NTBC, there exists significant compliance issues with the requirement for life-long daily dosing, and despite NTBC treatment continued concerns for the development of hepatocarcinoma exist.
  • TPP Target Product Profile
  • the drug target is the HPD gene, which encodes the enzyme that catalyzes the second step in the Tyr degradation process, upstream of the production of FAA.
  • HPD inactivation the therapeutic mechanism of our drug product, will prevent the accumulation of the toxic FAA and downstream metabolites including SA, which is used for diagnostic and surveillance purposes.
  • Liver and renal function is normal in patients with the ultrarare primary HPD deficiency, though more robust studies are needed to assess the full range of neurocognitive outcomes in untreated HPD deficiency patients 14,35 .
  • HuH-7 cells are imperfect proxies for authentic human hepatocytes. Accordingly, we are using primary human hepatocytes in parallel. Both in vitro models are critical for assessing the drug product for on-target HPD editing, effects on HPD protein expression, and off-target editing in the human genome. In order to assess for activity of therapeutic leads in vivo, we need an animal model that harbors the human HPD gene.
  • a humanized HPD mouse model (HPDhu/hu) is used as it will allow us to test the in vivo efficacy of human HPD gRNAs. However, HPDhu/hu mice will have the wild-type Fah gene and will not have HT1 disease.
  • HPDhu/hu/Fah–/– mice by breeding HPDhu/hu mice with homozygous Fah1RTyrc/RJ mice (The Jackson Laboratory #018129), which possess a point mutation in the Fah gene resulting in FAH deficiency and HT1 disease. The latter is the model in is used in the Examples above.
  • adenine base editing for precise disruption of a splice donor or acceptor site in the human HPD gene.
  • In utero adenine base editing to inactivate the Hpd gene rescues the lethal phenotype in the HT1 mouse model
  • the pathology of HT1 begins before birth and, if untreated, infants can present within weeks to months of birth with liver failure.
  • base editng technology was injected via the vitelline vein, which is akin to the umbilical vein in humans and drains directly into the portal circulation, to gestation day (E) 16 HT1 fetuses.
  • E gestation day
  • E16 Balb/c fetuses nondiseased fetuses
  • On-target hepatocyte baseline editing was between 5-15% depending on the delivery approach (AAV or LNP) (Fig. 18A).
  • EXAMPLE 10 In utero adenine base editing to inactivate the Hpd gene in the nonhuman primate model using the clinical HPD20 sgRNA
  • sgRNAs to target splice acceptor and splice donor sites in the human HPD gene to have 100% homology to the sequence in the nonhuman primate (NHP model.
  • This design allowed us to perform preclinical testing in a NHP model to be directly relevant to clinical translation.
  • Pritchard AB Izumi K, Payan-Walters I, Yudkoff M, Rand EB and Bhoj E. Inborn error of metabolism patients after liver transplantation: Outcomes of 35 patients over 27 years in one pediatric quaternary hospital. Am J Med Genet A. 2022;188:1443-1447 7. Hansen K and Horslen S. Metabolic liver disease in children. Liver Transpl. 2008;14:713- 733 8. Russo PA, Mitchell GA and Tanguay RM. Tyrosinemia: a review. Pediatr Dev Pathol. 2001;4:212-221 9.
  • In utero adenine base editing corrects multi-organ pathology in a lethal lysosomal storage disease. Nat Commun. 2021;12:4291.PMC8277817 19.
  • Gaudelli NM Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI and Liu DR. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature. 2017;551:464-471.PMC5726555 43.
  • Point mutations in the murine fumarylacetoacetate hydrolase gene Animal models for the human genetic disorder hereditary tyrosinemia type 1. Proc Natl Acad Sci U S A. 2001;98:641-645.PMC14641 51. Sun MS, Hattori S, Kubo S, Awata H, Matsuda I and Endo F. A mouse model of renal tubular injury of tyrosinemia type 1: development of de Toni Fanconi syndrome and apoptosis of renal tubular cells in Fah/Hpd double mutant mice. J Am Soc Nephrol. 2000;11:291-300 52. Jakobs C, Dorland L, Wikkerink B, Kok RM, de Jong AP and Wadman SK.

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Abstract

L'invention concerne des méthodes et des compositions pour des thérapies d'édition de base destinées au traitement de la tyrosinémie héréditaire de type 1 (TH1). Plus spécifiquement, l'invention concerne des méthodes et des compositions pour l'inactivation d'enzymes dans la voie catabolique de la tyrosine (Tyr) in vivo ou in utero pour empêcher l'accumulation de dérivés de fumarylaacétonacétate hydrolase (FAH) toxiques.
PCT/US2024/011693 2023-01-13 2024-01-16 Compositions et méthodes pour le traitement de la tyrosinémie héréditaire de type 1 WO2024152058A1 (fr)

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WO2021207710A2 (fr) * 2020-04-09 2021-10-14 Verve Therapeutics, Inc. Édition de base de angptl3 et procédés d'utilisation de celle-ci pour le traitement d'une maladie
WO2023281248A1 (fr) * 2021-07-05 2023-01-12 Ucl Business Ltd Traitement de troubles associés à la dégradation de la tyrosine

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021207710A2 (fr) * 2020-04-09 2021-10-14 Verve Therapeutics, Inc. Édition de base de angptl3 et procédés d'utilisation de celle-ci pour le traitement d'une maladie
WO2023281248A1 (fr) * 2021-07-05 2023-01-12 Ucl Business Ltd Traitement de troubles associés à la dégradation de la tyrosine

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