IP Library › Granted Patent US 12,636,381
Granted Patent B2
US 12,636,381 · App. 17/052,170 · Granted May 26, 2026

In utero CRISPR-mediated therapeutic editing of genes

Inventors: Kiran Musunuru (Philadelphia, PA); William H. Peranteau (Philadelphia, PA); Edward Morrisey (Newton Square, PA)
Assignees: THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA; THE CHILDREN'S HOSPITAL OF PHILADELPHIA
A61K48/005A61P3/00C12N9/0069C12N9/22C12N15/113C12N15/86C12N2310/11C12N2310/20C12N2710/10343C12N2750/14143C12Y113/11027
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Quick Facts
Patent No.
US 12,636,381
App. No.
17/052,170
Granted
May 26, 2026
Kind
B2
Abstract

A method for in utero genome editing, the method comprising administering to a subject an adenoviral vector, wherein the subject is a fetus, the adenoviral vector comprising CRISPR-mediated base editor and a guide RNA (gRNA), the gRNA targeting a mutation in a therapeutic gene; 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, an adeno-associated viral vector or lipid based nanoparticle.

Claims (26)

1 . A method for in utero genome editing in a fetal subject in need thereof, the method comprising:

(a) administering to the fetal subject, a pharmaceutical composition comprising at least one of

i) a lipid nanoparticle delivery vehicle;

ii) an adenovirus (AV) vector; or

iii) an adeno-associated virus (AAV) vector,

wherein at least one of i), ii) or iii) comprise nucleic acids encoding a CRISPR-mediated base editor 3 (BE3) or base editor 4 (BE4) and a guide RNA (gRNA) comprising a protospacer adjacent motif (PAM), which upon expression in said subject, form a CRISPR complex at a target site present in a 4-hydroxyphenylpyruvate dioxygenase (Hpd) gene; and

(b) introducing a modified codon at said target site in the Hpd gene by CRISPR complex mediated base editing for reducing Hpd expression, thereby treating, or reducing the risk of, hereditary tyrosinemia type I (HT1) disorder in said fetal subject, wherein said gRNA includes the nucleotide sequence of SEQ ID NO: 36, 5′-CATTCAACGTCACAACCACC-3′, an AGG PAM sequence and the editing is a C to T change on a sense strand encoding the Hpd gene at a mean editing rate of ˜15%.

2 . The method of claim 1 , further comprising before step (a):

(i) identifying a target codon for base editing into a nonsense codon; and

(ii) 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.

3 . The method of claim 2 , wherein 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.

4 . The method of claim 3 , further comprising assessing C bases within the window for a change to another base, wherein

a) the change is via a C to T or G to A on sense strand, and the modified codon is changed to a nonsense codon, or

b) the change is via a C to T or G to A on an antisense strand, and the modified codon is changed to a nonsense codon.

5 . The method of claim 4 , wherein the BE3 PAM sequence (NGG) in selected gRNAs is 13-17 nucleotides distal to the target cytosine base(s).

6 . The method of claim 1 , wherein the base editing occurs prior to onset of said disorder and said pharmaceutical composition is administered via injection into a vein in a human fetus.

7 . The method of claim 6 , wherein the base editing occurs in the liver of said fetal subject, wherein the fetal subject is inside a uterus of a body of a living carrier.

8 . The method of claim 1 , wherein the base editing decreases a risk of developing said disorder.

9 . The method of claim 6 , wherein the modified codon is a nonsense codon in the Hpd gene which knocks out Hpd gene function.

10 . The method of claim 9 , wherein loss of Hpd function restores normal liver function in the subject prior to birth or treats hereditary tyrosinemia type I (HT1) disorder in the subject prior to birth.

11 . The method of claim 1 , wherein said vector is an adeno-associated vector.

12 . The method of claim 1 , wherein said CRISPR-mediated base editor 3 (BE3) and a guide RNA (gRNA) are delivered on a lipid based nanoparticle.

13 . The method of claim 1 , wherein a combination of lipid-based nanoparticles and an AV vector are administered.

14 . The method of claim 1 , wherein a combination of lipid-based nanoparticles and an AAV vector are administered.

15 . The method of claim 1 , wherein said AAV vector is AAV8 or AAV9.

16 . The method of claim 1 , wherein said CRISPR-mediated base editor 4 (BE4) and a guide RNA (gRNA) are delivered on a lipid based nanoparticle.

Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 6, 2024
From: MUSUNURU, KIRAN; MORRISEY, EDWARD
To: THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA
Reel/Frame 066666/0567 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 27, 2021
From: PERANTEAU, WILLIAM
To: THE CHILDREN'S HOSPITAL OF PHILADELPHIA
Reel/Frame 057308/0769 →
Continuity (2)
Provisional Application 62664904 · Apr 30, 2018
Related Publication 20240075164A1 · Mar 7, 2024
References Cited (65)
US 4186183A · Steck et al. · 1980 [cited by applicant]
US 4217344A · Vanlerberghe et al. · 1980 [cited by applicant]
US 4235871A · Papahadjopoulos et al. · 1980 [cited by applicant]
US 4261975A · Fullerton et al. · 1981 [cited by applicant]
US 4485054A · Mezei et al. · 1984 [cited by applicant]
US 4501728A · Geho et al. · 1985 [cited by applicant]
US 4774085A · Fidler · 1988 [cited by applicant]
US 4797368A · Carter et al. · 1989 [cited by applicant]
US 4837028A · Allen · 1989 [cited by applicant]
US 4873316A · Meade et al. · 1989 [cited by applicant]
US 4897355A · Eppstein et al. · 1990 [cited by applicant]
US 4946787A · Eppstein et al. · 1990 [cited by applicant]
US 5049386A · Eppstein et al. · 1991 [cited by applicant]
US 5173414A · Lebkowski et al. · 1992 [cited by applicant]
US 20110059502A1 · Chalasani · 2011 [cited by applicant]
US 20160145646A1 · Frendewey et al. · 2016 [cited by applicant]
US 20160346362A1 · Quake et al. · 2016 [cited by applicant]
WO 1991016024A1 · 1991 [cited by applicant]
WO 1991017424A1 · 1991 [cited by applicant]
WO 1993024641A2 · 1993 [cited by applicant]
WO WO2017201258A1 · 2017 [cited by examiner]
Chadwick AC, Wang X, Musunuru K. In Vivo Base Editing of PCSK9 (Proprotein Convertase Subtilisin/Kexin Type 9) as a Therapeutic Alternative to Genome Editing. Arterioscler Thromb Vasc Biol. Sep. 2017;37(9):1741-1747. do… [cited by examiner]
Pierre Billon, Eric E. Bryant, Sarah A. Joseph, Tarun S. Nambiar, Samuel B. Hayward, Rodney Rothstein, Alberto Ciccia, CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of S… [cited by examiner]
Komor, A., Kim, Y., Packer, M. et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420-424 (2016). https://doi.org/10.1038/nature17946 (Year: 2016). [cited by examiner]
Lipshutz, Gerald S., Linda Flebbe-Rehwaldt, and Karin ML Gaensler. “Reexpression following readministration of an adenoviral vector in adult mice after initial in utero adenoviral administration.” Molecular Therapy 2.4 … [cited by examiner]
Pankowicz FP, Barzi M, Legras X, Hubert L, Mi T, Tomolonis JA, Ravishankar M, Sun Q, Yang D, Borowiak M, Sumazin P, Elsea SH, Bissig-Choisat B, Bissig KD. Reprogramming metabolic pathways in vivo with CRISPR/Cas9 genome… [cited by examiner]
NCBI Reference Sequence: NM_008277.3, Mus musculus 4-hydroxyphenylpyruvic acid dioxygenase (Hpd), mRNA, Oct. 7, 2017 revision (Year: 2017). [cited by examiner]
Waddington et al. In Utero gene therapy: current challenges and perspectives. Molecular Therapy. vol. 11, Issue 5, p. 661-676. May 2005. (Year: 2005). [cited by examiner]
Billon et al. Mol Cell . Sep. 21, 2017;67(6):1068-1079.e4. doi: 10.1016/j.molcel.2017.08.008. Epub Sep. 7, 2017. (Year: 2017). [cited by examiner]
Komor AC, Zhao KT, Packer MS, Gaudelli NM, Waterbury AL, Koblan LW, Kim YB, Badran AH, Liu DR. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficie… [cited by examiner]
Jia et al. Multiple sgRNAs facilitate base editing-mediated i-stop to induce complete and precise gene disruption. Protein Cell 2019, 10(11):832-839. (Year: 2019). [cited by examiner]
International Search Report and Written Opinion, dated Sep. 19, 2019, issued in corresponding International Application No. PCT/US2019/030083, filed Apr. 30, 2019. [cited by applicant]
Chadwick, Alexandra C. et al., “In Vivo Base Editing of PCSK9 (Proprotein Convertase Subtilisin/Kexin Type 9) as a Therapeutic Alternative to Genome Editing,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 37,… [cited by applicant]
Carreras, Alba et al., “In vivo genome and base editing of a human PCSK9 knock-in hypercholesterolemic mouse model,” BMC Biology, vol. 17, No. 4, Dec. 2019, pp. 1-14. [cited by applicant]
Kaufman, R.J. et al., “Translational efficiency of polycistronic mRNAs and their utilization to express heterologous genes in mammalian cells,” The EMBO Journal, vol. 6, No. 1, Jan. 1987, pp. 187-193. [cited by applicant]
Pinkert, Carl A. et al., “An albumin enhancer located 10 kb upstream functions along with its promoter to direct efficient, liver-specific expression in transgenic mice,” Genes & Development, vol. 1, 1987, pp. 268-276. [cited by applicant]
Calame, Kathryn et al., “Transcriptional Controlling Elements in the Immunoglobulin and T Cell Receptor Loci,” Advances in Immunology, vol. 43, 1988, pp. 235-275. [cited by applicant]
Winoto, Astar et al., “A novel, inducible and T cell-specific enhancer located at the 3′ end of the T cell receptor α locus,” The EMBO Journal, vol. 8, No. 3, Mar. 1989, pp. 729-733. [cited by applicant]
Banerji, J. et al., “A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes,” Cell, vol. 33, No. 3, Jul. 1983, pp. pp. 729-740. [cited by applicant]
Queen, C. et al., “Immunoglobulin gene transcription is activated by downstream sequence elements,” vol. 33, No. 3, Jul. 1983, pp. pp. 741-748. [cited by applicant]
Byrne, G.W. et al., “Multiplex gene regulation: A two-tiered approach to transgene regulation in transgenic mice,” PNAS, vol. 86, Jul. 1989, pp. 5473-5477. [cited by applicant]
Edlund, Thomas et al., “Cell-Specific Expression of the Rat Insulin Gene: Evidence for Role of Two Distinct 5′ Flanking Elements,” Science, vol. 230, No. 4728, Nov. 1985, pp. 912-916. [cited by applicant]
Kessel, M. et al., “Murine developmental control genes,” Science, vol. 249, No. 4967, Jul. 1990, pp. 374-379. [cited by applicant]
Camper, Sally A. et al., “Postnatal repression of the α-fetoprotein gene is enhancer independent,” Genes & Development, vol. 3, 1989, pp. 537-546. [cited by applicant]
Nakamura, Yasukazu et al., Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucleic Acids Research, vol. 28, No. Jan. 1, 2000, p. 292. [cited by applicant]
Anderson, W.F., “Human gene therapy,” Science, vol. 256, No. 5058, May 1992, pp. 808-813. [cited by applicant]
Miller, A.D., “Human gene therapy comes of age,” Nature, vol. 357, No. 6378, Jun. 1992, pp. 455-460. [cited by applicant]
Van Brunt, Jennifer, “Molecular Farming: Transgenic Animals as Bioreactors,” Biotechnology, vol. 6, No. 10, Oct. 1988, pp. 1149-1154. [cited by applicant]
Kremer, E.J. et al., “Adenovirus and adeno-associated virus mediated gene transfer,” British Medical Bulletin, vol. 51, No. 1, Jan. 1995, pp. 31-44. [cited by applicant]
Yu, M. et al., “Progress towards gene therapy for HIV infection,” Gene Therapy, vol. 1, No. 1, Jan. 1994, pp. 13-26. [cited by applicant]
Crystal, R.G., “Transfer of genes to humans: early lessons and obstacles to success,” Science, vol. 270, No. 5235, Oct. 1995, pp. 404-410. [cited by applicant]
Behr, Jean-Paul, “Gene Transfer with Synthetic Cationic Amphiphiles: Prospects for Gene Therapy,” Bioconjugate Chemistry, vol. 5, No. 5, Sep. 1994, pp. 382-389. [cited by applicant]
Remy, Jean-Serge et al., “Gene Transfer with a Series of Lipophilic DNA-Binding Molecules,” Bioconjugate Chemistry, vol. 5, No. 6, Nov. 1994, pp. 647-654. [cited by applicant]
Gao, X. et al., “Cationic liposome-mediated gene transfer,” Gene Therapy, vol. 2, No. 10, Dec. 1995, pp. 710-722. [cited by applicant]
Ahmad, Imran et al., “Antibody-mediated Specific Binding and Cytotoxicity of Liposome-entrapped Doxorubicin to Lung Cancer Cells in Vitro,” Cancer Research, vol. 52, Sep. 1992, pp. 4817-4820. [cited by applicant]
Johann, S. V. et al., GLVR1, a receptor for gibbon ape leukemia virus, is homologous to a phosphate permease of Neurospora crassa and is expressed at high levels in the brain and thymus, Journal of Virology, vol. 66, No… [cited by applicant]
Wilson, Carolyn et al., “Formation of infectious hybrid virions with gibbon ape leukemia virus and human T-cell leukemia virus retroviral envelope glycoproteins and the gag and pol proteins of Moloney murine leukemia vi… [cited by applicant]
Miller, A. Dusty et al., “Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus,” Journal of Virology, vol. 65, No. 5, May 1991, pp. 2220-2224. [cited by applicant]
West, Michael H. P. et al., “Gene expression in adeno-associated virus vectors: The effects of chimeric mRNA structure, helper virus, and adenovirus VA, RNA,” Virology, vol. 160, No. 1, Sep. 1987, pp. 38-47. [cited by applicant]
Kotin, Robert M., “Prospects for the Use of Adeno-Associated Virus as a Vector for Human Gene Therapy,” Human Gene Therapy, vol. 5, No. 7, Jul. 1994, pp. 793-801. [cited by applicant]
Muzyczka, Nicholas, “Adeno-associated Virus (AAV) Vectors: Will They Work?” Journal of Clinical Investigation, vol. 94, Oct. 1994, p. 1351. [cited by applicant]
Tratschin, Jon-Duri et al., “Adeno-associated virus vector for high-frequency integration, expression, and rescue of genes in mammalian cells,” Molecular and Cellular Biology, vol. 5, No. 11, Nov. 1985, pp. 3251-3260. [cited by applicant]
Tratschin, Jon-Duri et al., A human parvovirus, adeno-associated virus, as a eucaryotic vector: transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase, Molecular and Cellula… [cited by applicant]
Hermonat, Paul L. et al., “Use of adeno-associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells,” PNAS, vol. 81, No. 20, Oct. 1984, pp. 6466-6470. [cited by applicant]
Samulski, Richard Jude et al. “Helper-free stocks of recombinant adeno-associated viruses: normal integration does hot require viral gene expression,” Journal of Virology, vol. 63, No. 9, Sep. 1989, pp. 3822-3828. [cited by applicant]