IP Library › Granted Patent US 12,473,568
Granted Patent B2
US 12,473,568 · App. 19/064,383 · Granted Nov 18, 2025

Non-immunogenic circular, non-viral DNA vectors

Inventors: Jeffrey S. Bartlett (Columbus, OH); Ming Yan (Encino, CA)
Assignee: Rampart Bioscience, Inc.
C12N15/85A61K9/5123A61K48/0066C12N2750/14143C12N2800/107C12N2820/55
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Quick Facts
Patent No.
US 12,473,568
App. No.
19/064,383
Granted
Nov 18, 2025
Kind
B2
Abstract

The present disclosure relates to circular, non-viral DNA vectors, compositions including one or more of the disclosed vectors, and methods for delivering and/or expressing one or more therapeutic genes (e.g., proteins) in mammals, e.g., human patients. In some embodiments, the present disclosure is directed to circular, non-viral DNA vectors, such as circular non-viral DNA vectors including at least two inverted repeat sequences, where the at least two inverted repeat sequences are separated by a non-repeated nucleotide sequence which is not part of the at least two inverted repeat sequences. In some embodiments, the disclosed circular, non-viral DNA vectors do not include a “DD element.” In some embodiments, the disclosed circular, non-viral DNA vectors do not include a “DD element,” but include at least a portion of a bacterial origin of replication.

Claims (44)

1 . A pharmaceutical composition for administration to a human comprising:

(i) a circular, non-viral, non-integrating DNA vector comprising:

(a) a first portion comprising an expression cassette comprising a nucleic acid sequence encoding a therapeutic protein, wherein the nucleic acid sequence encoding the therapeutic protein is operably linked to a promoter; and

(b) a second portion comprising a nucleic acid sequence comprising a first inverted repeat sequence, a bacterial origin of replication (Ori) sequence, and a second inverted repeat sequence;

wherein the first inverted repeat sequence, bacterial Ori sequence, and second inverted repeat sequence are contiguous, and wherein the Ori sequence is flanked by, and in between, the first inverted repeat sequence and the second inverted repeat sequence; and

(ii) a lipid nanoparticle.

2 . The pharmaceutical composition of claim 1 , wherein the Ori sequence is derived from R6K.

3 . The pharmaceutical composition of claim 1 , wherein the Ori sequence is derived from an oriR6Kγ sequence.

4 . The pharmaceutical composition of claim 3 , wherein the oriR6Kγ sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 58, SEQ ID NO: 59, a nucleic acid sequence at least 90% identical to SEQ ID NO: 58, and a nucleic acid sequence at least 90% identical to SEQ ID NO: 59.

5 . The pharmaceutical composition of claim 1 , wherein the first inverted repeat sequence and second inverted repeat sequence are derived from an inverted repeat sequence from an adeno-associated virus (AAV).

6 . The pharmaceutical composition of claim 5 , wherein each of the inverted repeat sequences is independently at least 90% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-18.

7 . The pharmaceutical composition of claim 6 , wherein each of the inverted repeat sequences is independently selected from the group consisting of SEQ ID NOS: 1-18.

8 . The pharmaceutical composition of claim 1 , wherein the first inverted repeat sequence is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, and 17, and wherein the second inverted repeat is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, and 18.

9 . The pharmaceutical composition of claim 8 , wherein the first inverted repeat sequence is selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, and 17, and wherein the second inverted repeat sequence is selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, and 18.

10 . A pharmaceutical composition for administration to a human comprising:

(i) a circular, non-viral, non-integrating DNA vector comprising:

(a) a first portion comprising an expression cassette comprising a nucleic acid sequence encoding a therapeutic protein, wherein the nucleic acid sequence encoding the therapeutic protein is operably linked to a promoter; and

(b) a second portion comprising a nucleic acid sequence capable of forming a cruciform structure, wherein the nucleic acid sequence capable of forming a cruciform structure comprises the Formula X—Y—X′;

wherein X and X′ are each inverted repeat sequences and Y is a bacterial Ori sequence; and

(ii) a lipid nanoparticle.

11 . The pharmaceutical composition of claim 10 , wherein the Ori sequence is derived from R6K.

12 . The pharmaceutical composition of claim 10 , wherein the Ori sequence is derived from an oriR6Kγ sequence.

13 . The pharmaceutical composition of claim 12 , wherein the oriR6Kγ sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 58, SEQ ID NO: 59, a nucleic acid sequence at least 90% identical to SEQ ID NO: 58, and a nucleic acid sequence at least 90% identical to SEQ ID NO: 59.

14 . The pharmaceutical composition of claim 10 , wherein X and X′ are derived from an inverted repeat sequence from an AAV.

15 . The pharmaceutical composition of claim 14 , wherein X and X′ are independently at least 90% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-18.

16 . The pharmaceutical composition of claim 15 , wherein X and X′ are independently selected from the group consisting of SEQ ID NOS: 1-18.

17 . The pharmaceutical composition of claim 10 , wherein X is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, and 17, and wherein X′ is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, and 18.

18 . The pharmaceutical composition of claim 17 , wherein X is selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, and 17, and wherein X′ is selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, and 18.

19 . A pharmaceutical composition for administration to a human comprising:

(i) a circular, non-viral, non-integrating DNA vector comprising:

(a) a first portion comprising an expression cassette comprising a nucleic acid sequence encoding a therapeutic protein, wherein the nucleic acid sequence encoding the therapeutic protein is operably linked to a promoter; and

(b) a second portion comprising a nucleic acid sequence capable of forming a cruciform structure, wherein the nucleic acid sequence capable of forming a cruciform structure consists essentially of, operably linked in a 5′ to a 3′ direction:

(I) a first inverted repeat sequence;

(II) a bacterial Ori; and

(III) a second inverted repeat sequence; and

(ii) a lipid nanoparticle.

20 . The pharmaceutical composition of claim 19 , wherein the Ori is derived from R6K.

21 . The pharmaceutical composition of claim 19 , wherein the Ori is derived from an oriR6Kγ sequence .

22 . The pharmaceutical composition of claim 21 , wherein the oriR6Kγ sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 58, SEQ ID NO: 59, a nucleic acid sequence at least 90% identical to SEQ ID NO: 58, and a nucleic acid sequence at least 90% identical to SEQ ID NO: 59.

23 . The pharmaceutical composition of claim 19 , wherein each of the inverted repeat sequences is derived from an inverted repeat sequence from an AAV.

24 . The pharmaceutical composition of claim 23 , wherein each of the inverted repeat sequences is independently at least 90% identical to a sequence selected from the group consisting of SEQ ID NOS: 1-18.

25 . The pharmaceutical composition of claim 24 , wherein each of the inverted repeat sequences is independently selected from the group consisting of SEQ ID NOS: 1-18.

26 . The pharmaceutical composition of claim 19 , wherein the first inverted repeat sequence is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, and 17, and wherein the second inverted repeat is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, and 18.

27 . The pharmaceutical composition of claim 26 , wherein the first inverted repeat sequence is selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, and 17, and wherein the second inverted repeat sequence is selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, and 18.

Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 10, 2025
From: BARTLETT, JEFFREY S.
To: RAMPART BIOSCIENCE, INC.
Reel/Frame 072208/0134 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 10, 2025
From: YAN, MING
To: RAMPART BIOSCIENCE, INC.
Reel/Frame 072208/0163 →
Continuity (4)
Continuation 19015381 · Jan 9, 2025
Continuation PCTUS2023070238 · Jul 14, 2023
Provisional Application 63390515 · Jul 19, 2022
Related Publication 20250188487A1 · Jun 12, 2025
References Cited (132)
US 5478745A · Samulski et al. · 1995 [cited by applicant]
US 5869305A · Samulski et al. · 1999 [cited by applicant]
US 6977174B2 · Crouzet · 2005 [cited by examiner]
US 7763712B2 · Crine et al. · 2010 [cited by applicant]
US 7803622B2 · Engelhardt et al. · 2010 [cited by applicant]
US 7960529B2 · Crine et al. · 2011 [cited by applicant]
US 8691208B2 · Tomatsu et al. · 2014 [cited by applicant]
US 9598703B2 · Garcia et al. · 2017 [cited by applicant]
US 9988620B2 · Crine et al. · 2018 [cited by applicant]
US 10000532B2 · Crine et al. · 2018 [cited by applicant]
US 10047365B2 · Williams · 2018 [cited by applicant]
US 10167478B2 · Williams · 2019 [cited by applicant]
US 10590434B2 · Martin Molina et al. · 2020 [cited by applicant]
US 10822596B2 · Hatch · 2020 [cited by applicant]
US 10844388B2 · Williams · 2020 [cited by applicant]
US 11015210B2 · Rabinowitz · 2021 [cited by applicant]
US 11098313B2 · Williams · 2021 [cited by applicant]
US 11149302B2 · Rothwell et al. · 2021 [cited by applicant]
US 11324839B2 · Schnepp et al. · 2022 [cited by applicant]
US 11602569B2 · Schnepp et al. · 2023 [cited by applicant]
US 11634742B2 · de Beer et al. · 2023 [cited by applicant]
US 11766490B2 · Schnepp et al. · 2023 [cited by applicant]
US 11851665B2 · Williams · 2023 [cited by applicant]
US 20030032092A1 · Blanche et al. · 2003 [cited by applicant]
US 20080305073A1 · Barone et al. · 2008 [cited by applicant]
US 20100233814A1 · Williams · 2010 [cited by applicant]
US 20110250694A1 · Williams · 2011 [cited by applicant]
US 20200069817A1 · Liu et al. · 2020 [cited by applicant]
US 20210002667A1 · Schnepp et al. · 2021 [cited by applicant]
US 20210010021A1 · Williams · 2021 [cited by applicant]
US 20210169994A1 · Voegtli et al. · 2021 [cited by applicant]
US 20210269828A1 · Samulski · 2021 [cited by applicant]
US 20210330817A1 · Schnepp et al. · 2021 [cited by applicant]
US 20220090129A1 · Miller et al. · 2022 [cited by applicant]
US 20220098633A1 · De Beer et al. · 2022 [cited by applicant]
US 20220243201A1 · Maghodia et al. · 2022 [cited by applicant]
US 20220356490A1 · Liu et al. · 2022 [cited by applicant]
US 20240092905A1 · Sevy et al. · 2024 [cited by applicant]
US 20250146015A1 · Bartlett et al. · 2025 [cited by applicant]
US 20250146016A1 · Bartlett et al. · 2025 [cited by applicant]
US 20250179517A1 · Bartlett et al. · 2025 [cited by applicant]
EP 0828519B1 · 2008 [cited by applicant]
EP 3768846B1 · 2023 [cited by applicant]
WO WO9928340A2 · 1999 [cited by applicant]
WO WO9960146A1 · 1999 [cited by applicant]
WO WO2005084180A2 · 2005 [cited by applicant]
WO WO2009025866A1 · 2009 [cited by applicant]
WO WO2014035457A1 · 2014 [cited by applicant]
WO WO2014077863A1 · 2014 [cited by applicant]
WO WO2014077866A1 · 2014 [cited by applicant]
WO WO2014100798A1 · 2014 [cited by applicant]
WO WO2016025884A1 · 2016 [cited by applicant]
WO WO2018127462A1 · 2018 [cited by applicant]
WO WO2018213828A1 · 2018 [cited by applicant]
WO WO2019057774A1 · 2019 [cited by applicant]
WO WO2019143885A1 · 2019 [cited by applicant]
WO WO2019183248A1 · 2019 [cited by applicant]
WO WO2020033863A1 · 2020 [cited by applicant]
WO WO2020217057A1 · 2020 [cited by applicant]
WO WO2021022327A1 · 2021 [cited by applicant]
WO WO2021058984A1 · 2021 [cited by applicant]
WO WO2021119218A1 · 2021 [cited by applicant]
WO WO2022023284A1 · 2022 [cited by applicant]
WO WO2022051555A2 · 2022 [cited by applicant]
WO WO2022058755A1 · 2022 [cited by applicant]
WO WO2022087494A1 · 2022 [cited by applicant]
WO WO2022103852A1 · 2022 [cited by applicant]
WO WO2022147573A1 · 2022 [cited by applicant]
WO WO2022223556A1 · 2022 [cited by applicant]
WO WO2022271965A2 · 2022 [cited by applicant]
WO WO2023028441A1 · 2023 [cited by applicant]
WO WO2023028455A1 · 2023 [cited by applicant]
WO WO2020132396A1 · 2023 [cited by applicant]
WO WO2023056070A2 · 2023 [cited by applicant]
WO WO2023060088A1 · 2023 [cited by applicant]
WO WO2023122303A2 · 2023 [cited by applicant]
WO WO2023135273A2 · 2023 [cited by applicant]
WO WO2023178294A2 · 2023 [cited by applicant]
WO WO2024020320A2 · 2024 [cited by applicant]
WO WO2025090786A1 · 2025 [cited by applicant]
Lusby et al., “Nucleotide Sequence of the Inverted Terminal Repetition in Adeno-Associated Virus DNA” 34(2) Journal of Virology 402-409 (Year: 1980). [cited by examiner]
Nafissi et al., “Construction and Characterization of an in-vivo Linear Covalently Closed DNA Vector Production System” 11 Microbial Cell Factories 154, 1-13 (Year: 2012). [cited by examiner]
Bansal et al., “Non-canonical DNA structures: Diversity and disease association.” Frontiers in Genetics 13: 959258, Sep. 6, 2022. [cited by applicant]
Bochman et al., “DNA secondary structures: stability and function of G-quadruplex structures.” Nature Reviews/Genetics, p. 1-11, Oct. 3, 2012. [cited by applicant]
Bowater et al., “Interaction of Proteins with Inverted Repeats and Cruciform Structures in Nucleic Acids.” Int. J. Mol. Sci., 23, 6171, 2022. [cited by applicant]
Brazda et al., “Cruciform structures are common DNA feature important for regulating biological processes.” BMC Mol. Biol., 12:33, 2011. [cited by applicant]
Buck et al., “Recombinant Adeno-Associated viral Vectors (rAAV)-Vector Elements in Ocular Gene Therapy Clinical Trials and Transgene Expression and Bioactivity Assays.” Intl. J. of Mol. Sciences, 21, 4197, 2020. [cited by applicant]
Choi et al., “Effects of Adeno-Associated Virus DNA Hairpin Structure on Recombination.” J. of Virol., p. 6801-6807; vol. 79, No. 11, Jun. 2005. [cited by applicant]
Duan et al., “Circular Intermediates of Recombinant Adeno-Associated Virus Have Defined Structural Characteristics Responsible for Long-Term Episomal Persistence in Muscle Tissue.” J. of Virol., p. 8568-8577, Nov. 1998. [cited by applicant]
Durymanov et al., “Non-viral Delivery of Nucleic Acids: Insight Into Mechanisms of Overcoming Intracellular Barriers.” Front. Pharmacol. 9:971, Aug. 2021. [cited by applicant]
Earley et al., “Adeno-Associated Virus Serotype-Specific Inverted Terminal Repeat Sequence Role in Vector Transgene Expression.” Hum Gene Ther., 31(3-4):151-162, Feb. 2020. [cited by applicant]
Faust et al., CpG-depleted adeno-associated virus vectors evade immune detection. J Clin Invest, 123:2994-3001, 2013. [cited by applicant]
Finer et al., “A brief account of viral vectors and their promise for gene therapy.” Gene Ther., 24(1):1-2), 2017. [cited by applicant]
Goncalves et al., “DNA Minicircles Connected via G-Quadruplex Interaction Modules.” Small, 6, No. 12, 1347-1352, 2010. [cited by applicant]
Hardee et al., “Advances in Non-15 Viral DNA Vectors for Gene Therapy.” Genes 8(2), 65, 2017. [cited by applicant]
Hodgson et al., “Recent advances in non-viral vectors for gene therapy & vaccination.” Cell Gene Therapy Insights, 3(2), 95-101, 2017. [cited by applicant]
Inagaki et al., “Chromosomal instability mediated by non-B DNA: Cruciform confirmation and not DNA sequence is responsible for recurrent translocation in humans.” Genome Research 19:191-198; 2009. [cited by applicant]
Islas et al., “Design of microaerobically inducible miniR1 plasmids.” mLife, vol. 2, Issue 1; 101-104, Mar. 2023. [cited by applicant]
Kay et al., “A robust system for production of minicircle DNA vectors.” Nat. Biotechnol., 28, 1287-1289, 2010. [cited by applicant]
Kurahashi et al., “Cruciform DNA Structure Underlies the Etiology for Palindrome-mediated Human Chromosomal translocations.” J Biol Chem., 279(34):35377-35383, Aug. 20, 2004. [cited by applicant]
Li et al., “Production and characterization of novel recombinant adeno-associated virus replicative-form genomes: A eukaryotic source of DNA for gene transfer.” PLoS ONE 8, e69879, 2013. [cited by applicant]
Li et al., “Interaction In Vitro of Type III Intermediate Filament Proteins with Supercoiled Plasmid DNA and Modulation of Eukaryotic DNA Topoisomerase I and II Activities.” DNA and Cell Biology vol. 21, No. 10, p. 743-… [cited by applicant]
Lim et al., High spontaneous integration rates of end-modified linear DNAs upon 5 mammalian cell transfection. Sci Rep 13, 6835, 2023. [cited by applicant]
Loot et al., “Cellular pathways controlling integron cassette site folding.” EMBO, 29, 2623-2634, 2010. [cited by applicant]
Lu et al., “The extragenic spacer length between the 5′ and 3′ ends of the transgene expression cassette affects transgene silencing from plasmid-based vectors.” Mol. Ther. J. Am. Soc. Gene Ther., 20, 2111-2119, 2012. [cited by applicant]
Maniar et al., “Minicircle DNA Vectors Achieve Sustained Expression Reflected by Active Chromatin and Transcriptional Level.” Mol. Ther., 21, 131-138, 2013. [cited by applicant]
Mayer et al., “Construction of DNA Architecture with RNA Hairpins.” Angw. Chem. Int. Ed., 47, 971-973, 2008. [cited by applicant]
McLean et al., “The Role of DNA Sequence in the Formation of Z-DNA Versus Cruciforms in Plasmids.” J. Biol. Chem. Vol. 263, No. 15, pp. 7370-7377, May 25, 1988. [cited by applicant]
Nielsen et al., “A reproducible method for identification of human genomic DNA autonomously replicating sequences.” Mol. Gen. Genet., 242:280-288, 1994. [cited by applicant]
Ou-Yang et al., “Cruciform DNA Structures Act as Legible Templates for Accelerating Homologous Recombination in Transgenic Animals.” Int. J. Mol. Sci., 23, 3973, 2022. [cited by applicant]
Pan et al., “Rational engineering of a functional CpG-free ITR for AAV gene therapy.” Gene Ther., 29(6): 333-345, Jun. 2022. [cited by applicant]
Pandya et al., “Regulatory role of Non-canonical DNA Polymorphisms in human genome and their relevance in Cancer.” BBA-Reviews on Cancer 1876, 188594, 2021. [cited by applicant]
Pierce et al., “High throughput Single-Cell Chromatin Accessibility CRISPR Screens Enable Unbiased Identification of Regulatory Networks in Cancer.” Nature Communications, 12:2969, May 2021. [cited by applicant]
Puras et al., “Protamine/DNA/Niosome Ternary Nonviral Vectors for Gene Delivery to the Retina: The Role of Protamine.” Mol. Pharm., 12, 3658-3671, 2015. [cited by applicant]
Rasched et al., “DNA Minicircles with Gaps for Versatile Functionalization.” Angw. Chem. Int. Ed., 47, 967-970, 2008. [cited by applicant]
Samulski et al., “AAV-mediated gene therapy for research and therapeutic purposes.” Annu. Rev. 5 Virol. 1, 427-451, 2014. [cited by applicant]
Samulski et al., Cloning of adeno-associated virus into pBR322: Rescue of intact virus from the recombinant plasmid in human cells.: PNAS USA vol. 79, pp. 2077-2081, Mar. 1982. [cited by applicant]
Sarkar et al., “A novel approach to design of cis-acting DNA structural elements for regulation of gene expression in vivo.” Current Science, Vil. 60, Nos. 9 & 10, 25, p. 586-591, May 1991. [cited by applicant]
Schnepp et al., “Infectious Molecular Clones of Adeno-Associated Virus Isolated Directly from Human Tissues.” J. of Virol., p. 1456-1464, vol. 83, No. 3, Feb. 2009. [cited by applicant]
Thibault et al., “Production of DNA minicircles less than 250 base pairs through a novel concentrated DNA circularization assay enabling minicircle design with NF-kB inhibition activity.” Nucleic Acid Research, vol. 45,… [cited by applicant]
Tolmachov, O.E. “Self-entanglement of long linear DNA vectors using transient non-B-DNA attachment points: A new concept for improvement of non-viral therapeutic gene delivery.” Medical Hypotheses 78, 632-635, 2012. [cited by applicant]
Van Gaal et al., “Plasmid Engineering for Controlled and Sustained Gene Expression for Nonviral Gene Therapy.” Pharm. Res., 23, 1053-1074, 2006. [cited by applicant]
Williams, J.A., “Vector Design for Improved DNA Efficacy, Safety and Production.” Vaccines, 1, 224-249, 2013. [cited by applicant]
Williams et al., “Improving cell and gene therapy safety and performance using next-generation Nanoplasmid vectors.” Molecular Therapy: Nucleic Acids vol. 32, p. 494-503, Jun. 2023. [cited by applicant]
Xiao et al., “A Novel 165-Base Pair Terminal Repeat Sequence is the Sole CIS Requirement for the Adeno-Associated Virus Life Cycle.” J. of Virol., vol. 71, No. 2, p. 941-948, Feb. 1997. [cited by applicant]
Zhang et al., “Letter to the Editor: ”D“ matters in recombinant AAV packaging.” Mol. Ther. vol. 29, No. 6, p. 1937-1939, Jun. 2021. [cited by applicant]
Zhao et al., “Viral vector-based gene therapies in the clinic.” Bioeng Transl Med., 7(1): 30 e10258, Jan. 2022. [cited by applicant]
International Search Report and Written Opinion of PCT/US23/70238, dated Feb. 13, 2024. [cited by applicant]
U.S. Appl. No. 19/015,381, filed Jan. 9, 2025, Bartlett, et al. [cited by applicant]
U.S. Appl. No. 19/016,927, filed Jan. 10, 2025, Bartlett, et al. [cited by applicant]
U.S. Appl. No. 19/060,511, filed Feb. 21, 2025, Bartlett, et al. [cited by applicant]
Athman et al., “A Computational Analysis of Alternative Splicing Across Mammalian Tissues Reveals Circadian and Ultradian Rhythms in Splicing Events” pp. 1-25, International Journal of Molecular Sciences, Aug. 15, 2019. [cited by applicant]