IP Library › Granted Patent US 12,600,984
Granted Patent B2
US 12,600,984 · App. 17/026,101 · Granted Apr 14, 2026

Viral and non-viral nanoplasmid vectors with improved production

Inventor: James A. Williams (Lincoln, NE)
Assignee: Aldevron, L.L.C.
C12N15/85C12N15/70C12N15/86C12N2740/15043C12N2740/15052C12N2750/14143C12N2750/14152C12N2800/90C12N2820/55
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Quick Facts
Patent No.
US 12,600,984
App. No.
17/026,101
Granted
Apr 14, 2026
Kind
B2
Abstract

A method for improving the replication of a covalently closed circular plasmid is provided. The method includes providing a covalently closed circular plasmid having a Pol I-dependent origin of replication, and an insert including a structured DNA sequence selected from inverted repeat sequences, direct repeat sequences, homopolymeric repeat sequences, eukaryotic origins of replication or eukaryotic promoter enhancer sequences, wherein the structured DNA sequence is located at a distance of less than 1000 bp from the Pol I-dependent origin of replication in the direction of replication. The method also includes modifying the covalently closed circular recombinant molecule such that the Pol I-dependent origin of replication is replaced with a Pol III-dependent origin of replication, whereby the resultant Pol III-dependent origin of replication covalently closed circular plasmid has improved replication. An antibiotic marker free covalently closed circular recombinant DNA molecule is also provided.

Claims (35)

1 . A method for replicating a covalently closed circular plasmid comprising the following steps:

a. providing a cell containing a covalently closed circular plasmid, the covalently closed circular plasmid comprising:

i. a backbone comprising a Pol III-dependent R6K origin of replication and an RNA-OUT RNA selectable marker, wherein the backbone is less than 1000 bp, and

ii. an insert comprising a structured DNA sequence selected from the group consisting of an inverted repeat sequence, a direct repeat sequence, and a eukaryotic origin of replication, wherein the structured DNA sequence is located at a distance of less than 1000 bp from the Pol III-dependent R6K origin of replication in the direction of replication; and

b. subjecting the cell to a fermentation process.

2 . The method of claim 1 , wherein the cell is an engineered Rep protein-expressing E. coli strain.

3 . The method of claim 2 , wherein the cell comprises a chromosomally-integrated arabinose inducible CI857ts gene.

4 . The method of claim 3 , wherein Rep protein includes the following mutations: P42L; P106I; F107S; and P113S.

5 . The method of claim 1 , wherein the insert is a transposon vector.

6 . The method of claim 5 , wherein the transposon vector comprises an inverted repeat sequence, or a direct repeat sequence.

7 . The method of claim 1 , wherein the insert is a transposase vector.

8 . The method of claim 1 , wherein the insert is a viral vector and the viral vector is an AAV vector.

9 . The method of claim 8 , wherein the AAV vector comprises an inverted repeat sequence.

10 . The method of claim 1 , wherein the insert is a viral vector and the viral vector is a lentiviral vector.

11 . The method of claim 10 , wherein the lentiviral vector comprises a direct repeat sequence or a eukaryotic origin of replication.

12 . The method of claim 1 , wherein the Pol III-dependent R6K origin of replication possesses at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 18.

13 . The method of claim 1 , wherein the RNA-OUT RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 5, and SEQ ID NO: 7.

14 . The method of claim 1 , wherein the structured DNA sequence is selected from the group consisting of a SV40 origin of replication, a viral LTR, a Lentiviral LTR, a Retroviral LTR, a transposon IR/DR repeat, a Sleeping Beauty transposon IR/DR repeat, and an AAV ITR.

15 . The method of claim 1 , wherein the fermentation process comprises growing the cells in media containing arabinose.

16 . The method of claim 1 , wherein the cell is an engineered Rep protein-expressing E. coli strain and comprises a chromosomally-integrated arabinose inducible CI857ts gene, wherein the structured DNA sequence is selected from the group consisting of a SV40 origin of replication, a viral LTR, a Lentiviral LTR, a Retroviral LTR, a transposon IR/DR repeat, a Sleeping Beauty transposon IR/DR repeat, and an AAV ITR, wherein the Pol III-dependent R6K origin of replication possesses at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 18, wherein the RNA-OUT RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 5, and SEQ ID NO: 7.

17 . The method of claim 16 , wherein the yield of the covalently closed circular plasmid following the fermentation process is in excess of 0.5 g/L.

18 . A covalently closed circular plasmid comprising a backbone and an insert, the backbone comprising a Pol III-dependent R6K origin of replication and an RNA-OUT RNA selectable marker, wherein the backbone is less than 1000 bp, and the insert comprises a structured DNA sequence selected from the group consisting of an inverted repeat sequence, a direct repeat sequence, and a eukaryotic origin of replication, wherein the structured DNA sequence is located at a distance of less than 1000 bp from the Pol III-dependent R6K origin of replication in the direction of replication.

19 . The covalently closed circular plasmid of claim 18 , wherein the Pol III-dependent R6K origin of replication possesses at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 18.

20 . The covalently closed circular plasmid of claim 18 , wherein the RNA-OUT RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 5, and SEQ ID NO: 7.

21 . The covalently closed circular plasmid of claim 18 , wherein the structured DNA sequence is selected from the group consisting of a SV40 origin of replication, a viral LTR, a Lentiviral LTR, a Retroviral LTR, a transposon IR/DR repeat, a Sleeping Beauty transposon IR/DR repeat, and an AAV ITR.

22 . The covalently closed circular plasmid of claim 18 , wherein the insert is a transposon vector.

23 . The covalently closed circular plasmid of claim 22 , wherein the transposon vector comprises an inverted repeat sequence, or a direct repeat sequence.

24 . The covalently closed circular plasmid of claim 18 , wherein the insert is a transposase vector.

25 . The covalently closed circular plasmid of claim 18 , wherein the insert is a viral vector and the viral vector is an AAV vector.

26 . The covalently closed circular plasmid of claim 25 , wherein the AAV vector comprises an inverted repeat sequence.

27 . The covalently closed circular plasmid of claim 18 , wherein the insert is a viral vector and the viral vector is a lentiviral vector.

28 . The covalently closed circular plasmid of claim 27 , wherein the lentiviral vector comprises a direct repeat sequence or a eukaryotic origin of replication.

29 . The covalently closed circular plasmid of claim 18 , wherein the Pol III-dependent R6K origin of replication possesses at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 18, and wherein the RNA-OUT RNA selectable marker is an RNA-IN regulating RNA-OUT functional variant with at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 5, and SEQ ID NO: 7.

30 . The method of claim 1 , wherein replication of the covalently closed circular plasmid occurs without production of replication intermediates.

31 . The covalently closed circular plasmid of claim 18 , wherein the covalently closed circular plasmid is configured to replicate without production of replication intermediates.

Assignments (2)
MERGER Recorded Feb 22, 2023
From: NATURE TECHNOLOGY CORPORATION
To: ALDEVRON, L.L.C.
Reel/Frame 062770/0971 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 17, 2020
From: WILLIAMS, JAMES A.
To: NATURE TECHNOLOGY CORPORATION
Reel/Frame 054086/0964 →
Continuity (3)
Continuation PCTUS2019023209 · Mar 20, 2019
Provisional Application 62645892 · Mar 21, 2018
Related Publication 20210010021A1 · Jan 14, 2021
References Cited (103)
US 5922583A · Morsey · 1999 [cited by applicant]
US 6709844B1 · Levy · 2004 [cited by applicant]
US 6977174B2 · Crouzet et al. · 2005 [cited by applicant]
US 7244609B2 · Drocourt et al. · 2007 [cited by applicant]
US 7611883B2 · Cranenburgh · 2009 [cited by applicant]
US 7943377B2 · Carnes et al. · 2011 [cited by applicant]
US 9012226B2 · Williams · 2015 [cited by applicant]
US 9017966B2 · Williams et al. · 2015 [cited by applicant]
US 9018012B2 · Williams · 2015 [cited by applicant]
US 9506082B2 · Williams · 2016 [cited by applicant]
US 9550998B2 · Williams · 2017 [cited by applicant]
US 9950081B2 · Williams · 2018 [cited by examiner]
US 20030180949A1 · Levy · 2003 [cited by applicant]
US 20040072326A1 · Cayota Guzicovsky · 2004 [cited by examiner]
US 20060063232A1 · Grabherr et al. · 2006 [cited by applicant]
US 20090298909A1 · Pachuk · 2009 [cited by applicant]
US 20100184158A1 · Williams · 2010 [cited by applicant]
US 20130344604A1 · Aebischer-Gumy · 2013 [cited by applicant]
US 20150191735A1 · Williams · 2015 [cited by examiner]
US 20150275221A1 · Williams · 2015 [cited by applicant]
US 20160008488A1 · Williams · 2016 [cited by examiner]
CA 2794446 · 2002 [cited by applicant]
CN 1665930 · 2005 [cited by applicant]
CN 101932708 · 2010 [cited by applicant]
EP 13100166A1 · 2003 [cited by applicant]
EP 1642970A1 · 2006 [cited by applicant]
EP 1921140A1 · 2008 [cited by applicant]
JP H04197185A · 1992 [cited by applicant]
JP H11502705A · 1999 [cited by applicant]
JP 2016521553A · 2016 [cited by applicant]
JP 2021518150A · 2019 [cited by applicant]
WO 9630531A · 1996 [cited by applicant]
WO WO9630531A1 · 1996 [cited by examiner]
WO 2002013602A · 2002 [cited by applicant]
WO WO0213602A1 · 2002 [cited by examiner]
WO 2004033664A2 · 2004 [cited by applicant]
WO 2005003342A · 2005 [cited by applicant]
WO WO2005003342A1 · 2005 [cited by examiner]
WO 2007004642A · 2007 [cited by applicant]
WO WO2007004642A1 · 2007 [cited by examiner]
WO 2008153733A2 · 2008 [cited by applicant]
WO 2014035457A1 · 2014 [cited by applicant]
WO 2014077863A1 · 2014 [cited by applicant]
WO 2014077866A1 · 2014 [cited by applicant]
WO 2014195920A2 · 2014 [cited by applicant]
WO 2017025447A1 · 2017 [cited by applicant]
WO 2017066579A1 · 2017 [cited by applicant]
WO 2020132396A1 · 2020 [cited by applicant]
Carnes et al. ECI Digital Archives, Vaccine Technology VI, Jun. 12, 2016 (Year: 2016). [cited by examiner]
Lu et al. (Human Gene Therapy, 2016, 28(1), p. 125-134, Supplementary Figure S2) (Year: 2016). [cited by examiner]
Carnes, A., Tiwari, N., Beilowitz, J., Sampson, C., Peterson, D. and Williams, J., 2016. Production of a Nanoplasmid™ with a large gene insert using the HyperGRO™ fermentation process. ECI Digital Archives, Vaccine Tech… [cited by examiner]
Williams, S.L., Casas-Delucchi, C.S., Raguseo, F., Guneri, D., Li, Y., Minamino, M., Fletcher, E.E., Yeeles, J.T., Keyser, U.F., Waller, Z.A. and Di Antonio, M., 2023. Replication-induced DNA secondary structures drive … [cited by examiner]
Office Action mailed Dec. 29, 2023 in Chinese Patent Application No. 201980034026.9. [cited by applicant]
Office Action mailed Jan. 9, 2024 in Japanese Patent Application No. 2020-551263. [cited by applicant]
Official Action issued on Mar. 14, 2023, in Japanese Patent Application No. 2020-551263. [cited by applicant]
Carnes A et al, Production of a Nanoplasmid with a large gene insert using the HyperGRO fermentation process, Engineering Conferences International ECI Digital Archives, Vaccine Technology VI2016. [cited by applicant]
European Search Report issued on Jan. 25, 2023, in European Patent Application No. 22194460.6. [cited by applicant]
Office Action (and English translation) dated Aug. 8, 2023 for Japanese Patent Application No. 2020-551263. [cited by applicant]
Abhyankar et al.; Reconstitution of R6K Dna Replication in Vitro Using 22 Purified Proteins; 2003 J Biol Chem 278:45476-45484. [cited by applicant]
Abhyankar et al.; Biochemical Investigations of Control of Replication Initiation of Plasmid R6K; 2004 J Biol Chem 279:6711-6719. [cited by applicant]
Allen et al.; Roles of DNA polymerase I in leading and lagging-strand replication defined by a high-resolution mutation footprint of ColE1 plasmid replication; 2011 Nucleic Acids Research 39:7020-33. [cited by applicant]
Carnes et al.; Critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust RNA Pol II and Pol III-mediated eukaryotic expression with high bacterial production yields; J Gene Medicin… [cited by applicant]
Carnes A.E.; Fermentation Design for the Manufacture of Therapeutic Plasmid DNA; 2005 BioProcess Intl 3:36-44. [cited by applicant]
Chadeuf et al.; Evidence for Encapsidation of Prokaryotic Sequences during Recombinant Adeno-Associated Virus Production and Their in Vivo Persistence after Vector Delivery; 2005 Molecular Therapy, vol. 12, No. 4:744-75… [cited by applicant]
Chenna et al.; Evidence for Encapsidation of Prokaryotic Sequences during Recombinant Adeno-Associated Virus Production and Their in Vivo Persistence after Vector Delivery; 2003 Nucleic Acids Res., vol. 31, No. 13:3497-… [cited by applicant]
Chen ZY, He CY, Meuse L, Kay MA; Silencing of episomal transgene expression by plasmid bacterial DNA elements in vivo; 2004 Gene Ther 11:856-864. [cited by applicant]
Del Solar et al.; Replication and Control of Circular Bacterial Plasmids; 1998 Microbiol. Mol. Biol. Rev vol. 62, No. 2:434-464. [cited by applicant]
Elango et al.; Optimized transfection of mRNA transcribed from a d(A/T)100 tail-containing vector; Biochemical and Biophysical Research Communications 330(2005):958-966. [cited by applicant]
Franch T, and Gerdes K.; U-turns and regulatory RNAs; 2000. Current Opin Microbiol 3:159-164. [cited by applicant]
Godiska, Ronald & Patterson, Melodee & Schoenfeld, Tom & Mead, David. (2005). Mini Review Beyond pUC: Vectors for Cloning Unstable Dna. Dna Sequencing: Optimizing the Process and Analysis; pp. 55-75. [cited by applicant]
Hebel HL, Cai Y, Davies LA, Hyde SC, Pringle IA, Gill DR; Challenges in the Process Development of a Novel Zero CpG CFTR Plasmid for Human Clinical Use; 2008 Molecular Therapy; vol. 16, Supp. 1: S110. [cited by applicant]
Holstein et al.; Efficient Non-viral Gene Delivery into Human Hematopoietic Stem Cells by Minicircle Sleeping Beauty Transposon Vectors; (2018) Molecular Therapy epub Jan. 17, 2018. [cited by applicant]
Karbownickez et al.; Doggybone DNA: an advanced platform for AAV production; 2017. Cell and Gene Therapy Insights 731-738. [cited by applicant]
Kay, Mark; et al.; A Robust System for Production of Minicircle DNA Vectors; Dec. 2010 Nat Biotechnol 28(12):1287-1289. [cited by applicant]
Krasilnikova, et al.; Transcription through a simple DNA repeat blocks replication elongation; EMBO Journal; v. 17; No. 17; pp. 5095-5102 (1998). [cited by applicant]
Lu J., et al.; The Extragenic Spacer Length Between the 5′ and 3′ Ends of the Transgene Expression Cassette Affects Transgene Silencing from Plasmid-based Vectors; 2012 Mol Ther. vol. 20, No. 11:2111-2119. [cited by applicant]
Lu, J., et al.: “A 5′ Noncoding Exon Containing Engineered Intron Enhances Trangene Expression from Recombinant AAV Vectors in Vivo”, Human Gene Therapy, vol. 28, No. 1, Jan. 1, 2017 (Jan. 1, 2017), pp. 125-134. [cited by applicant]
Luke et al.; Vector Insert-Targeted Integrative Antisense Expression System for Plasmid Stabilization; 2011 Mol Biotechnol 47:43. [cited by applicant]
Marie et al.; pFARs, Plasmids free of antibiotic resistance markers, display high-level transgene expression in muscle, skin and tumour cells; (2010) J. Gene Medicine 12:323-332. [cited by applicant]
Marino MP, Luce MJ, Reiser J.; Small- to Large-Scale Production of Lentivirus Vectors; 2003, Methods Mol Biol 229:43-55. [cited by applicant]
Mirkin and Mirkin; Replication Fork Stalling at Natural Impediments; 2007 Microbiology and Molecular Biology Reviews 71:13-35. [cited by applicant]
Monjezi, et al.; Enhanced CAR T-cell engineering using non-viral Sleeping Beauty transposition from minicircle vectors; (2017) Leukemia 31:186-194. [cited by applicant]
Mutalik et al.; Rationally designed families of orthogonal RNA regulators of translation; 2012 Nat Chem Biol 8:447-454. [cited by applicant]
Na D., et al.; Metabolic engineering of Escherichia coli using synthetic small regulatory NRAs; 2013 Nat Biotechnol 31:170-174. [cited by applicant]
Pastor, M., et al.; The Antibiotic-free pFAR4 Vector Paired with the Sleeping Beauty Transposon System Mediates Efficient Transgene Delivery in Human Cells; 2018 Molecular Therapy 11:57-67. [cited by applicant]
Quiviger, M., et al.; High and prolonged sulfamidase secretion by the liverof MPS-IIIA mice following hydrodynamic tail vein delivery of antibiotic-free pFAR4 plasmid vector; 2014 Gene Therapy 21:1001-1007. [cited by applicant]
Ribeiro et al., Plasmid DNA Size Does Affect Nonviral Gene Delivery Efficiency in Stem Cells; 2011 Cell Reprogram vol. 14, No. 2:130. [cited by applicant]
Schnodt et al., DNA Minicircle Technology Improves Purity of Adeno-associated Viral Vector Preparations; 2016 Mol Ther - Nucleic Acids 5 e355. [cited by applicant]
Sharma et al.; Efficient Sleeping Beauty DNA Transposition From DNA Minicircles; 2013 Molecular Therapy Nucleic Acids 2:e74. [cited by applicant]
Soubrier, et al.; pCOR: a new design of plasmid vectors for nonviral gene therapy; 1999 Gene Therapy 6:1482-1488. [cited by applicant]
Suzuki M., et al.; Plasmid DNA Sequences Present in Conventional Herpes Simplex Virus Amplicon Vectors Cause Rapid Transgene Silencing by Forming Inactive Chromatin; 2006. J Virol 80(7):3293-3300. [cited by applicant]
Tipanee, J., et al.; Preclinical and clinical advances in transposon-based gene therapy; 2017 Bioscience reports, 37(6), BSR20160614. https://doi.org/10.1042/BSR20160614. [cited by applicant]
Wagner, E., et al.; Antisense RNAs in Bacteria and Their Genetic Elements; 2002 Adv Genet 46:361-398. [cited by applicant]
Wang et al., In Vivo Electroporation of Minicircle DNA as a Novel Method of Vaccine Delivery To Enhance HIV-1- Specific Immune Responses; 2014 J Virology 88:1924-34. [cited by applicant]
Williams, J.A., et al.; Plasmid DNA Vaccine Vector Design: Impact on Efficacy, Safety, and Upstream Production; Biotechnology Advances, Elsevier Publishing, Barking, GB, vol. 27, No. 4, Jul. 1, 2009 (Jul. 1, 2009). [cited by applicant]
Wilson, I. W., et al.; Importance of Structural Differences between Complementary RNA Molecules to Control of Replication of an IncB Plasmid; 1997 J Bacteriol 179(3):742-53. [cited by applicant]
Wu, F., et al.; A DNA Segment Conferring Stable Maintenance on R6K y-Origin Core Replicons; 1995 J Bacteriol. 177(22): 6338-6345. [cited by applicant]
International Search Report and Written Opinion of related application PCT/US19/23209, dated May 22, 2019. [cited by applicant]
Office Action mailed Nov. 6, 2024 in Chinese Patent Application No. 201980034026.9. [cited by applicant]
Office Action mailed Nov. 25, 2024 in Canadian Patent Application No. 3,093,346. [cited by applicant]
Office Action mailed Oct. 30, 2024 in Korean Patent Application No. 10-2020-7030139. [cited by applicant]
Lu, et al., “Multi-origin usage for chromosome replication of suppressive integration strain of dnaA46 mutant of [cited by applicant]
Search Report mailed Dec. 29, 2023 in Chinese Patent Application No. 201980034026.9. [cited by applicant]