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EP-4736889-A2 - COMPOSITIONS AND METHODS FOR DELIVERY OF NUCLEIC ACIDS TO CELLS

EP4736889A2EP 4736889 A2EP4736889 A2EP 4736889A2EP-4736889-A2

Abstract

Compositions and methods of use thereof for delivering nucleic acid cargo into cells are provided. The compositions typically include (a) a 3E10 monoclonal antibody or an antigen binding, cell-penetrating fragment thereof; a monovalent, divalent, or multivalent single chain variable fragment (scFv); or a diabody; or humanized form or variant thereof, and (b) a nucleic acid cargo including, for example, a nucleic acid encoding a polypeptide, a functional nucleic acid, a nucleic acid encoding a functional nucleic acid, or a combination thereof. Elements (a) and (b) are typically non-covalently linked to form a complex.

Inventors

  • QUIJANO, Elias
  • GLAZER, PETER

Assignees

  • Yale University

Dates

Publication Date
20260506
Application Date
20200831

Claims (15)

  1. A composition comprising or consisting of (a) a monoclonal antibody, cell-penetrating fragment thereof; a monovalent, divalent, or multivalent single chain variable fragment (scFv); or a diabody; or humanized form or variant thereof, comprising (i) a first heavy chain CDR selected from any of SEQ ID NOS: 15 and 16 (as identified by Kabat), a second heavy chain CDR selected from any of SEQ ID NOS: 17, 19, and 42 (as identified by Kabat), and a third heavy chain CDR SEQ ID NO: 18 (as identified by Kabat); in combination with a first light chain CDR selected from any of SEQ ID NOS: 24, 27, 44 (as identified by Kabat), a second light chain CDR selected from any of SEQ ID NOS: 25 and 28 (as identified by Kabat), and a third light chain CDR comprising SEQ ID NO:26 (as identified by Kabat); or (ii) a first heavy chain CDR selected from any of SEQ ID NOS: 20 and 21 (according to IMGT), a second heavy chain CDR selected from any of SEQ ID NOS: 22 and 43 (according to IMGT), and a third heavy chain CDR SEQ ID NO: 23 (according to IMGT); in combination with a first light chain CDR selected from any of SEQ ID NOS: 29 and 45 (according to IMGT), a second light chain CDR SEQ ID NO: 30 (according to IMGT), and a third light chain CDR SEQ ID NO:26 (according to IMGT), and (b) a nucleic acid cargo comprising a nucleic acid encoding a polypeptide, a functional nucleic acid, a nucleic acid encoding a functional nucleic acid, or a combination thereof, wherein (a) and (b) are non-covalently linked.
  2. The composition of claim 1, wherein (a) is bispecific.
  3. The composition of claim 1 or 2, wherein (b) comprises DNA, RNA, PNA or other modified nucleic acids, or nucleic acid analogs, or a combination thereof, optionally wherein (b) comprises mRNA.
  4. The composition of any one of claims 1-3, wherein (b) comprises a vector, optionally wherein the vector comprises a nucleic acid sequence encoding a polypeptide of interest operably linked to an expression control sequence, optionally, wherein the vector is a plasmid.
  5. The composition of any one of claims 1-4, wherein (b) comprises a nucleic acid encoding a Cas endonuclease, a gRNA, or a combination thereof.
  6. The composition of any one of claims 1-5, wherein (b) comprises a nucleic acid encoding a chimeric antigen receptor polypeptide.
  7. The composition of any one of claims 1-6, wherein (b) comprises a functional nucleic acid or a nucleic acid encoding a functional nucleic acid.
  8. The composition of claim 7, wherein the functional nucleic acid is an antisense molecule, an siRNA, a miRNA, an aptamer, a ribozyme, an RNAi, or an external guide sequence.
  9. The composition of any one of claims 1-8, wherein (b) comprises a plurality of a single nucleic acid molecule.
  10. The composition of any one of claims 1-9, wherein (b) comprises or consists of single stranded nucleic acids, double stranded nucleic acids, or a combination thereof.
  11. The composition of any one of claims 1-10, further comprising carrier DNA, optionally wherein the carrier DNA is non-coding DNA.
  12. A pharmaceutical composition comprising the composition of any one of claims 1-11 and a pharmaceutically acceptable excipient.
  13. The composition of claim 12 further comprising polymeric nanoparticles encapsulating a complex of (a) and (b), optionally wherein a targeting moiety, a cell penetrating peptide, or a combination thereof is associated with, linked, conjugated, or otherwise attached directly or indirectly to the nanoparticle.
  14. A method of making the composition of any one of claims 1-13, comprising incubating and/or mixing of (a) and (b) for between about 1 min and about 30 min, about 10 min and about 20 min, or about 15 min, optionally at room temperature or 37 degrees Celsius.
  15. A composition or method of any one of claims 1-14 wherein the amino acid residue corresponding with D31 or N31 of a heavy chain amino acid sequence or a CDR thereof is substituted with R or L.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 62/944,281, entitled "Compositions And Methods For Delivery Of Nucleic Acids To Cells", filed in the United States Patent and Trademark Office on December 5, 2019, International Application No. PCT/US2019/048953, entitled "Compositions And Methods For Enhancing Donor Oligonucleotide-Based Gene Editing" and filed in the United States Receiving Office for the Patent Cooperation Treaty on August 30, 2019, and International Application No. PCT/US2019/048962, entitled "Compositions And Methods For Enhancing Triplex And Nuclease-Based Gene Editing" and filed in the United States Receiving Office for the Patent Cooperation Treaty on August 30, 2019. U.S. Provisional Application No. 62/944,281, International Application No. PCT/US2019/048953, U.S. Provisional Application No. 62/725,920, International Application No. PCT/US2019/048962, U.S. Provisional Application No. 62/725,852 are each specifically incorporated by reference in their entireties. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under CA197574 awarded by National Institutes of Health. The Government has certain rights in the invention. REFERENCE TO THE SEQUENCE LISTING The Sequence Listing submitted as a text file named "YU_7503_3_ST25" created on August 31, 2020, and having a size of 154,701 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5). FIELD OF THE INVENTION The invention is generally related to the field of intracellular delivery of nucleic acids, for application including, but not limited to in vitro, ex vivo, and in vivo gene therapy and gene editing. BACKGROUND OF THE INVENTION Gene therapy includes a spectrum of applications ranging from gene replacement and knockdown for genetic or acquired diseases such as cancer, to vaccination. Viral vectors and synthetic liposomes have emerged as the vehicles of choice for many applications today, but both have limitations and risks, including complexity of production, limited packaging capacity, and unfavorable immunological features, which restrict gene therapy applications and hold back the potential for preventive gene therapy (Seow and Wood, Mol Ther. 17(5): 767-777 (2009). In vivo uptake and distributed of nucleotide in cells and tissues has been observed (Huang, et al., FEBS Lett., 558(1-3):69-73 (2004)). Further, although, for example, Nyce, et al. have shown that antisense oligodeoxynucleotides (ODNs) when inhaled bind to endogenous surfactant (a lipid produced by lung cells) and are taken up by lung cells without a need for additional carrier lipids (Nyce, et al., Nature, 385:721-725 (1997)), small nucleic acids are taken up into T24 bladder carcinoma tissue culture cells (Ma, et al., Antisense Nucleic Acid Drug Dev., 8:415-426 (1998)), there remains a need for improved nucleic acid transfection technology, particularly for in vivo applications. AAV9, still the viral vector typically used in people was discovered in 2003 (Robbins, "Gene therapy pioneer says the field is behind - and that delivery technology is embarrassing," Stat, November, 2019). Thus, it is an object of the invention to provided compositions and methods of use thereof for improved delivery of nucleic acids into cells. SUMMARY OF THE INVENTION Compositions and methods of use thereof for delivering nucleic acid cargo into cells are provided. The compositions typically include (a) a 3E10 monoclonal antibody or a cell-penetrating fragment thereof; a monovalent, divalent, or multivalent single chain variable fragment (scFv); or a diabody; or humanized form or variant thereof, and (b) a nucleic acid cargo including, for example, a nucleic acid encoding a polypeptide, a functional nucleic acid, a nucleic acid encoding a functional nucleic acid, or a combination thereof. Elements (a) and (b) are typically non-covalently linked to form a complex. It is believed that in additional to DNA, 3E10 binds to RNA, PNA, and other nucleic acids. Exemplary 3E10 antibodies and fragments and fusion protein thereof include those having (i) the CDRs of any one of SEQ ID NO:1-6, 12, 13, 46-48, or 50-52 in combination with the CDRs of any one of SEQ ID NO:7-11, 14, or 53-58; (ii) first, second, and third heavy chain CDRs selected from SEQ ID NOS:15-23, 42, and 43 in combination with first, second and third light chain CDRs selected from SEQ ID NOS:24-30, 44, and 45; (iii) a humanized forms of (i) or (ii); (iv) a heavy chain comprising an amino acid sequence comprising at least 85% sequence identity to any one of SEQ ID NO:1 or 2 in combination with a light chain comprising an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO:7 or 8; (v) a humanized form or (iv); or (vi) a heavy chain comprising an amino acid sequence comprising at least 85% sequence identity to any one of SEQ ID NO:3-6, 46-48, or 50-52 in combination with a