The content of the electronically submitted sequence listing (Name: 4471_0070003_SequenceListing_ST26; Size: 211,617 Bytes; and Date of Creation: Mar. 29, 2024) is herein incorporated by reference in its entirety.
The present disclosure provides expression vectors, bacterial sequence-free vectors, vector production systems for making the bacterial sequence-free vectors, and uses thereof.
Gene therapy has significant therapeutic promise, but challenges remain in realizing its potential.
Most clinical trials have utilized viral delivery systems, such as adenoviral vectors, lentiviral vectors, and adeno-associated viral vectors. While progress has been made, viral systems vary in transduction and transgene expression efficiencies and concerns remain regarding undesirable effects such as inflammatory and immune responses or insertional mutagenesis. Moreover, production, purification, and storage of viral vectors is often costly, highly variable, and inefficient. See, e.g., Lingelbach, D., Drug Development & Delivery 20(5): 50-54 (2020); Wright, J. F., Gene Therapy 15:840-848 (2008).
Nonviral vectors also have been investigated as gene therapy delivery systems. While safer than their viral counterparts, the effectiveness of nonviral vectors can be limited, for example, by low transgene expression levels and durability of expression. See, e.g., Kay, M., Nature Reviews Genetics 12: 316-328 (2011).
There is a need for improved vectors such as those described herein.
The present disclosure is directed to an expression vector comprising: (a) a backbone sequence, (b) a sequence comprising: (i) an expression cassette comprising a nucleic acid sequence of interest, (ii) a first target sequence for a first recombinase flanking the 5′ side of the expression cassette, (iii) a second target sequence for the first recombinase flanking the 3′ side of the expression cassette, and (iv) one or more additional target sequences for one or more additional recombinases integrated within the first and second target sequences in non-binding regions for the first recombinase, and (c) one or more of: (i) an endonuclease target sequence integrated within the first and/or second target sequences for the first recombinase in non-binding regions for the first recombinase and the one or more additional recombinases, wherein the endonuclease target sequence is between the backbone sequence and cleavage sites for the first recombinase and the one or more additional recombinases, (ii) a synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:12 integrated between the 3′ end of the first target sequence for the first recombinase and the 5′ end of another enhancer or a promoter in the expression cassette, (iii) a cytomegalovirus (CMV) enhancer integrated between the 3′ end of the first target sequence for the first recombinase and the 5′ end of a promoter in the expression cassette, (iv) a 5′ untranslated region (5′UTR) comprising an intron, wherein the 5′UTR is integrated in the expression cassette between a promoter and the nucleic acid sequence of interest, (v) a vertebrate chromatin insulator integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal, (vi) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal, (vii) a scaffold/matrix attachment region (S/MAR) integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal, or (viii) a DNA nuclear targeting sequence (DTS) integrated within the first and/or second target sequences for the first recombinase in non-binding regions for the first recombinase and the one or more additional recombinases, wherein the DTS is between the expression cassette and cleavage sites for the first recombinase and the one or more additional recombinases.
In some aspects, the expression vector comprises an endonuclease target sequence integrated within the first and/or second target sequences for the first recombinase in non-binding regions for the first recombinase and the one or more additional recombinases, wherein the endonuclease target sequence is between the backbone sequence and cleavage sites for the first recombinase and the one or more additional recombinases. In some aspects, the endonuclease target sequence is integrated within the first and second target sequences for the first recombinase. In some aspects, the endonuclease target sequence is for a homing endonuclease. In some aspects, the endonuclease target sequence is for I-AniI, I-CeuI, I-ChuI, I-CpaI, I-CpaII, I-CreI, I-DmoI, H-DreI, I-HmuI, I-HmuII, I-LlaI, I-MsoI, PI-PfuI, PI-PkoII, I-PorI, I-PpoI, PI-PspI, I-ScaI, I-SceI, PI-SceI, I-SceII, I-SecIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-Ssp6803I, I-TevI, I-TevII, I-TevIII, PI-TliI, PI-TliII, I-Tsp061I, or I-Vdi141I. In some aspects, the endonuclease target sequence is for I-SceI. In some aspects, the endonuclease target sequence is for PI-SceI. In some aspects, the endonuclease target sequence is for a Cas endonuclease. In some aspects, the Cas endonuclease is Cas9.
In some aspects, the expression vector comprises a synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:12 integrated between the 3′ end of the first target sequence for the first recombinase and the 5′ end of another enhancer or a promoter in the expression cassette. In some aspects, the synthetic enhancer comprises multiple contiguous copies of a nucleic acid sequence at least about 90% identical to SEQ ID NO:12. In some aspects, the synthetic enhancer comprises a nucleic acid sequence at least about 90% identical to SEQ ID NO:46. In some aspects, the synthetic enhancer is integrated at the 5′ end of a chicken β-actin promoter. In some aspects, a chimeric intron comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:47 is integrated at the 3′ end of the chicken β-actin promoter and 5′ to the nucleic acid sequence of interest.
In some aspects, the expression vector comprises a CMV enhancer integrated between the 3′ end of the first target sequence for the first recombinase and the 5′ end of a promoter in the expression cassette. In some aspects, the CMV enhancer is integrated at the 3′ end of a synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:12 or SEQ ID NO:46. In some aspects, a CMV promoter is integrated at the 3′ end of the CMV enhancer and 5′ to the nucleic acid sequence of interest.
In some aspects, the expression vector comprises a nucleic acid sequence at least about 90% identical to SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39 integrated between the first target sequence for the first recombinase and the nucleic acid sequence of interest.
In some aspects, the expression vector comprises a 5′UTR comprising an intron, wherein the 5′UTR is integrated in the expression cassette between a promoter and the nucleic acid sequence of interest. In some aspects, the intron comprises a nucleic acid sequence at least about 90% identical to SEQ ID NO:1. In some aspects, the 5′UTR further comprises a non-coding sequence integrated within the intron. In some aspects, the 5′UTR comprises a non-coding sequence integrated between two of the nucleotides in the intron corresponding to any two nucleotides from positions 25 to 55 of SEQ ID NO:1. In some aspects, the non-coding sequence is an S/MAR. In some aspects, the S/MAR is MAR-5. In some aspects, the 5′UTR comprises a nucleic acid sequence at least about 90% identical SEQ ID NO:3. In some aspects, the 5′UTR comprises a nucleic acid sequence at least about 90% identical SEQ ID NO:5. In some aspects, the promoter is a chicken β-actin promoter. In some aspects, the promoter is a CMV promoter. In some aspects, the promoter is integrated at the 3′ end of a CMV enhancer. In some aspects, the CMV enhancer is integrated at the 3′ end of a synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO: 12 or SEQ ID NO:46.
In some aspects, the expression vector comprises a polyadenylation signal that is integrated at the 3′ end of the nucleic acid sequence of interest. In some aspects, the polyadenylation signal comprises a nucleic acid sequence at least about 90% identical to SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.
In some aspects, the expression vector comprises a vertebrate chromatin insulator integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal. In some aspects, the vertebrate chromatin insulator is 5′-HS4 chicken-β-globin insulator (cHS4). In some aspects, the polyadenylation signal comprises a nucleic acid sequence at least about 90% identical to SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.
In some aspects, the expression vector comprises a WPRE integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal. In some aspects, the polyadenylation signal comprises a nucleic acid sequence at least about 90% identical to SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
In some aspects, the expression vector comprises a S/MAR integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal. In some aspects, the S/MAR is MAR-5. In some aspects, the polyadenylation signal comprises a nucleic acid sequence at least about 90% identical to SEQ ID NO: 13, SEQ ID NO:14, or SEQ ID NO:15.
In some aspects, the expression vector comprises an enhancer sequence flanking each side of the first and second target sequences for the first recombinase. In some aspects, the expression vector comprises at least two enhancer sequences flanking each side of the first and second target sequences for the first recombinase. In some aspects, the enhancer sequence is a SV40 enhancer sequence.
In some aspects, the expression vector comprises a DTS integrated within the first and/or second target sequences for the first recombinase in non-binding regions for the first recombinase and the one or more additional recombinases, wherein the DTS is between the expression cassette and cleavage sites for the first recombinase and the one or more additional recombinases. In some aspects, the DTS is a SV40 enhancer sequence. In some aspects, the DTS is cell-specific.
In some aspects, the first and second target sequences and the one or more additional target sequences are selected from the group consisting of the PY54 pal site, the N15 telRL site, the loxP site, φK02 telRL site, the FRT site, the phiC31 attP site, and the λ attP site. In some aspects, the expression vector comprises each of the target sequences. In some aspects, the expression vector comprises the pal site and the telRL, loxP, and FRT recombinase target binding sequences integrated within the pal site. In some aspects, the first and second target sequences for the first recombinase each comprise the nucleic acid sequence of SEQ ID NO:33.
In some aspects, the expression vector is for producing a bacterial sequence-free vector. In some aspects, the bacterial sequence-free vector is a circular covalently closed vector. In some aspects, the bacterial sequence-free vector is a linear covalently closed vector.
The present disclosure is directed to a vector production system comprising recombinant cells encoding a recombinase under the control of an inducible promoter, wherein the recombinant cells comprise any of the above expression vectors, and wherein the recombinase targets the first and second target sequences for the first recombinase or one of the one or more additional target sequences for the one or more additional recombinases in the expression vector. In some aspects, the recombinase is TelN, Tel, Cre, or Flp.
In some aspects, the recombinant cells further encode an endonuclease under the control of an inducible promoter, wherein the endonuclease targets the endonuclease target sequence in an expression vector comprising the endonuclease target sequence. In some aspects, the endonuclease is a homing endonuclease. In some aspects, the homing endonuclease is I-AniI, I-CeuI, I-ChuI, I-CpaI, I-CpaII, I-CreI, I-DmoI, H-DreI, I-HmuI, I-HmuII, I-LlaI, I-MsoI, PI-PfuI, PI-PkoII, I-PorI, I-PpoI, PI-PspI, I-ScaI, I-SceI, PI-SceI, I-SceII, I-SecIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-Ssp6803I, I-TevI, I-TevII, I-TevIII, PI-TliI, PI-TliII, I-Tsp061I, or I-Vdi141I. In some aspects, the endonuclease is I-SceI. In some aspects, the endonuclease is PI-SceI. In some aspects, the recombinant cells encode a nuclease genome editing system comprising the endonuclease. In some aspects, the nuclease genome editing system is a clustered regularly interspaced short palindromic repeats (CRISPR) nuclease system comprising a guide RNA and a Cas endonuclease. In some aspects, the Cas endonuclease is Cas9. In some aspects, the inducible promoter is thermally-regulated, chemically-regulated, IPTG regulated, glucose-regulated, arabinose inducible, T7 polymerase regulated, cold-shock inducible, pH inducible, or combinations thereof.
The present disclosure is directed to a method of producing a bacterial sequence-free vector comprising incubating any of the above vector production systems under suitable conditions for expression of the recombinase. In some aspects, the method further comprises incubating any of the above vector production systems that encode an endonuclease under suitable conditions for expression of the endonuclease. In some aspects, the method further comprises incubating any of the above vector production systems that encode a nuclease genome editing system under suitable conditions for expression of the nuclease genome editing system. In some aspects, the method further comprises harvesting the bacterial sequence-free vector.
The present disclosure is directed to a bacterial sequence-free vector produced by any of the above methods of producing a bacterial sequence-free vector.
The present disclosure is directed to a bacterial sequence-free vector comprising: (a) an expression cassette comprising a nucleic acid sequence of interest, and (b) one or more of: (i) a synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:12 located 5′ to another enhancer or a promoter in the expression cassette, (ii) a CMV enhancer located 5′ to a promoter in the expression cassette, (iii) a 5′UTR comprising an intron, wherein the 5′UTR is integrated in the expression cassette between a promoter and the nucleic acid sequence of interest, (iv) a vertebrate chromatin insulator integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal, (v) a WPRE integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal, (vi) a S/MAR integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal, or (vii) a DTS located 5′ to the expression cassette.
In some aspects, the bacterial sequence-free vector comprises a synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:12 located 5′ to another enhancer or a promoter in the expression cassette. In some aspects, the synthetic enhancer comprises multiple contiguous copies of a nucleic acid sequence at least about 90% identical to SEQ ID NO: 12 In some aspects, the synthetic enhancer comprises a nucleic acid sequence at least about 90% identical to SEQ ID NO:46. In some aspects, the synthetic enhancer is integrated at the 5′ end of a chicken β-actin promoter. In some aspects, a chimeric intron comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:47 is integrated at the 3′ end of the chicken β-actin promoter and 5′ to the nucleic acid sequence of interest.
In some aspects, the bacterial sequence-free vector comprises a CMV enhancer located 5′ to a promoter in the expression cassette. In some aspects, the CMV enhancer is integrated at the 3′ end of a synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:12 or SEQ ID NO:46. In some aspects, a CMV promoter is integrated at the 3′ end of the CMV enhancer and 5′ to the nucleic acid sequence of interest.
In some aspects, the bacterial sequence-free vector comprises a nucleic acid sequence at least about 90% identical to SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39 located 5′ to the nucleic acid sequence of interest.
In some aspects, the bacterial sequence-free vector comprises a 5′UTR comprising an intron, wherein the 5′UTR is integrated in the expression cassette between a promoter and the nucleic acid sequence of interest. In some aspects, the intron comprises a nucleic acid sequence at least about 90% identical to SEQ ID NO:1. In some aspects, the 5′UTR further comprises a non-coding sequence integrated within the intron. In some aspects, the 5′UTR further comprises a non-coding sequence integrated between two of the nucleotides in the intron corresponding to any two nucleotides from nucleotide positions 25 and 55 of SEQ ID NO: 1. In some aspects, the non-coding sequence is an S/MAR. In some aspects, the S/MAR is MAR-5. In some aspects, the 5′UTR comprises a nucleic acid sequence at least about 90% identical SEQ ID NO:3. In some aspects, the 5′UTR comprises a nucleic acid sequence at least about 90% identical SEQ ID NO:5. In some aspects, the promoter is a chicken β-actin promoter. In some aspects, the promoter is a CMV promoter. In some aspects, the promoter is integrated at the 3′ end of a CMV enhancer. In some aspects, the CMV enhancer is integrated at the 3′ end of a synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO: 12 or SEQ ID NO:46.
In some aspects, the bacterial sequence-free vector comprises a polyadenylation signal that is integrated at the 3′ end of the nucleic acid sequence of interest. In some aspects, the polyadenylation signal comprises a nucleic acid sequence at least about 90% identical to SEQ ID NO:13, SEQ ID NO: 14, or SEQ ID NO:15.
In some aspects, the bacterial sequence-free vector comprises a vertebrate chromatin insulator integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal. In some aspects, the vertebrate chromatin insulator is cHS4. In some aspects, the polyadenylation signal comprises a nucleic acid sequence at least about 90% identical to SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
In some aspects, the bacterial sequence-free vector comprises a WPRE integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal. In some aspects, the polyadenylation signal comprises a nucleic acid sequence at least about 90% identical to SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.
In some aspects, the bacterial sequence-free vector comprises a S/MAR integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal. In some aspects, the S/MAR is MAR-5.
In some aspects, the bacterial sequence-free vector comprises an enhancer sequence flanking each side of the expression cassette. In some aspects, the bacterial sequence-free vector comprises at least two enhancer sequences flanking each side of the expression cassette. In some aspects, the enhancer sequence is a SV40 enhancer sequence.
In some aspects, the bacterial sequence-free vector comprises a DTS located 5′ to the expression cassette. In some aspects, the DTS is a SV40 enhancer sequence. In some aspects, the DTS is cell-specific.
In some aspects, the bacterial sequence-free vector is a circular covalently closed vector.
In some aspects, the bacterial sequence-free vector is a linear covalently closed vector.
The present disclosure is directed to a recombinant cell comprising any of the above expression vectors or any of the above bacterial sequence-free vectors.
The present disclosure is directed to a composition comprising any of the above expression vectors or any of the above bacterial sequence-free vectors. In some aspects, the composition further comprises a delivery agent. In some aspects, the delivery agent is a nanoparticle. In some aspects, the delivery agent comprises a targeting ligand. In some aspects, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
The present disclosure is directed to a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject any of the above expression vectors, any of the above bacterial sequence-free vectors, or the above pharmaceutical composition.
The present disclosure is directed to a polynucleotide comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:1.
The present disclosure is directed to a polynucleotide comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:2.
The present disclosure is directed to a polynucleotide comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:3.
The present disclosure is directed to a polynucleotide comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:5.
The present disclosure is directed to a polynucleotide comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO: 12.
The present disclosure is directed to a polynucleotide comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:46.
The present disclosure is directed to a polynucleotide comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:13.
The present disclosure is directed to a polynucleotide comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:14.
The present disclosure is directed to a polynucleotide comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:15.
In some aspects, any of the above polynucleotides comprising a nucleic acid sequence at least about 90% identical to any one of SEQ ID NOs: 13-15 further comprises 100 to 120 adenine nucleotides at the 3′ end of the nucleic acid sequence.
The present disclosure is directed to a polynucleotide comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:16.
The present disclosure is directed to a polynucleotide comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:17.
The present disclosure is directed to a polynucleotide comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:18.
The present disclosure is directed to a polynucleotide comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:35.
The present disclosure is directed to a polynucleotide comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:36.
The present disclosure is directed to a polynucleotide comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:37.
The present disclosure is directed to a polynucleotide comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:38.
The present disclosure is directed to a polynucleotide comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:39.
The present disclosure is directed to an expression vector comprising any of the above polynucleotides.
The present disclosure is directed to an expression vector comprising a polynucleotide comprising a nucleic acid sequence at least about 90% identical to any one of SEQ ID NOs:2, 3, or 5, and (i) a polynucleotide comprising a nucleic acid sequence at least about 90% identical to any one of SEQ ID NOs: 13-18, or (ii) a polynucleotide comprising a nucleic acid sequence at least about 90% identical to any one of SEQ ID NOs:13-15 and 100 to 120 adenine nucleotides at the 3′ end of the nucleic acid sequence.
The present disclosure is directed to a method of gene editing comprising inserting a nucleic acid sequence of interest from any of the above expression vectors, any of the bacterial sequence-free vectors, or any of the above pharmaceutical compositions into a target site for gene editing. In some aspects, the gene editing is by non-homologous end joining. In some aspects, the gene editing is by homology-directed repair.
The present disclosure provides expression vectors, bacterial sequence-free vectors (e.g., ministring DNA (msDNA)), vector production systems, methods of making the bacterial sequence-free vectors, compositions, and uses thereof.
All publications cited herein are hereby incorporated by reference in their entireties, including without limitation all journal articles, books, manuals, patent applications, and patents cited herein, to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.
It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a nucleotide sequence,” is understood to represent one or more nucleotide sequences. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
The term “and/or” where used herein is to be taken as specific disclosure of each of the specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of” and/or “consisting essentially of” are also provided.
The terms “about” or “comprising essentially of” refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” or “comprising essentially of” can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, “about” or “comprising essentially of” can mean a range of up to 10% (i.e., +10%). Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the application and claims, unless otherwise stated, the meaning of “about” or “comprising essentially of” should be assumed to be within an acceptable error range for that particular value or composition.
As described herein, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Numeric ranges are inclusive of the numbers defining the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 5th ed., 2013, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, 2006, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.
Units, prefixes, and symbols are denoted in their Système International de Unites (SI) accepted form.
Unless otherwise indicated, nucleotide sequences are written left to right in 5′ to 3′ orientation. Amino acid sequences are written left to right in amino to carboxy orientation.
The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.
“Amino acid” is a molecule having the structure wherein a central carbon atom (the alpha-carbon atom) is linked to a hydrogen atom, a carboxylic acid group (the carbon atom of which is referred to herein as a “carboxyl carbon atom”), an amino group (the nitrogen atom of which is referred to herein as an “amino nitrogen atom”), and a side chain group, R. When incorporated into a peptide, polypeptide, or protein, an amino acid loses one or more atoms of its amino acid carboxylic groups in the dehydration reaction that links one amino acid to another. As a result, when incorporated into a protein, an amino acid is referred to as an “amino acid residue.”
“Protein” or “polypeptide” refers to any polymer of two or more individual amino acids (whether or not naturally occurring) linked via a peptide bond, and occurs when the carboxyl carbon atom of the carboxylic acid group bonded to the alpha-carbon of one amino acid (or amino acid residue) becomes covalently bound to the amino nitrogen atom of amino group bonded to the non alpha-carbon of an adjacent amino acid. The terms “protein” and “polypeptide” can be used interchangeably herein. Similarly, fragments of proteins and polypeptides are also within the scope of the disclosure and may be referred to herein as “proteins” or “polypeptides.” In one aspect of the disclosure, a polypeptide comprises a chimera of two or more parental peptide segments or proteins. The term “polypeptide” is also intended to refer to and encompass the products of post-translation modification (“PTM”) of the polypeptide, including without limitation disulfide bond formation, glycosylation, carbamylation, lipidation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, modification by non-naturally occurring amino acids, or any other manipulation or modification, such as conjugation with a labeling component. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis. An “isolated” polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can simply be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the disclosure, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
Recombinant polypeptides (i.e., recombinant proteins) comprising two or more proteins as disclosed herein can be encoded by a single coding sequence that comprises polynucleotide sequences encoding each protein. Unless stated otherwise, the polynucleotide sequences encoding each protein are “in frame” such that translation of a single mRNA comprising the polynucleotide sequences results in a single polypeptide comprising each protein. Typically, the proteins in a recombinant polypeptide as described herein will be fused directly to one another or will be separated by a peptide linker. Various polynucleotide sequences encoding peptide linkers are known in the art and include, for example, self-cleaving peptides.
“Polynucleotide” or “nucleic acid” as used herein refers to a polymeric form of nucleotides. In some instances, a polynucleotide comprises a sequence that is either not immediately contiguous with the coding sequences or is immediately contiguous (on the 5′ end or on the 3′ end) with the coding sequences in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA) independent of other sequences. The nucleotides of the disclosure can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. A polynucleotide as used herein refers to, among others, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. The term polynucleotide encompasses genomic DNA or RNA (depending upon the organism, i.e., RNA genome of viruses), as well as mRNA encoded by the genomic DNA, and cDNA. In certain aspects, a polynucleotide comprises a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)). By “isolated” nucleic acid or polynucleotide is intended a nucleic acid molecule, e.g., DNA or RNA, which has been removed from its native environment. For example, a nucleic acid molecule comprising a polynucleotide encoding a recombinant polypeptide contained in a vector is considered “isolated” for the purposes of the present disclosure. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) from other polynucleotides in a solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present disclosure. Isolated polynucleotides or nucleic acids according to the present disclosure further include polynucleotides and nucleic acids (e.g., nucleic acid molecules) produced synthetically.
As used herein, a “coding region” or “coding sequence” is a portion of a polynucleotide, which consists of codons translatable into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is typically not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. The boundaries of a coding region are typically determined by a start codon at the 5′ terminus, encoding the amino-terminus of the resultant polypeptide, and a translation stop codon at the 3′ terminus, encoding the carboxyl-terminus of the resulting polypeptide.
As used herein, an “expression cassette” comprises a nucleic acid sequence of interest (e.g., a nucleic acid sequence for expression of a polypeptide, DNA, or RNA) and an expression control region.
As used herein, a “transgene” can be used interchangeably with “gene of interest (GOI)” and refers to a portion of a polynucleotide that contains codons translatable into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is typically not translated into an amino acid, it can be considered to be part of a transgene, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of the transgene. The boundaries of a transgene are typically determined by a start codon at the 5′ terminus, encoding the amino-terminus of the resultant polypeptide, and a translation stop codon at the 3′ terminus, encoding the carboxyl-terminus of the resulting polypeptide.
As used herein, the term “expression control region” refers to a transcription control element that is operably associated with a coding region to direct or control expression of the product encoded by the coding region, including, for example, cis-regulatory modules (CRMs), promoters (e.g., a tissue specific promoter and/or an inducible promoter), enhancers, operators, repressors, ribosome binding sites, translation leader sequences, introns, post-transcriptional elements, polyadenylation recognition sequences, RNA processing sites, effector binding sites, stem-loop structures, and transcription termination signals, miRNA binding sites, and combinations thereof. Expression control regions include nucleotide sequences located upstream (5′), within, or downstream (3′) of a nucleic acid sequence of interest, and which influence the transcription, RNA processing, stability, or translation of the associated nucleic acid sequence of interest. If a transgene is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3′ to the transgene.
A coding region and a promoter are “operably associated” (i.e., “operably linked”) if induction of promoter function results in the transcription of mRNA comprising a coding region that encodes the product, and if the nature of the linkage between the promoter and the coding region does not interfere with the ability of the promoter to direct the expression of the product encoded by the coding region or interfere with the ability of the DNA template to be transcribed. Expression control regions include nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding region, and which influence the transcription, RNA processing, stability, or translation of the associated coding region. If a coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence.
As used herein, the terms “host cell” and “cell” can be used interchangeably and can refer to any type of cell or a population of cells, e.g., a primary cell, a cell in culture, or a cell from a cell line, that harbors or is capable of harboring a nucleic acid molecule (e.g., a recombinant nucleic acid molecule). Host cells can be a prokaryotic cell, or alternatively, the host cells can be eukaryotic, for example, fungal cells, such as yeast cells, and various animal cells, such as insect cells or mammalian cells.
“Culture,” “to culture” and “culturing,” as used herein, means to incubate cells under in vitro conditions that allow for cell growth or division or to maintain cells in a living state. “Cultured cells,” as used herein, means cells that are propagated in vitro.
A “subject” includes any human or nonhuman animal. The term “nonhuman animal” includes, but is not limited to, vertebrates such as mammals, avians, pets, farm animals, nonhuman primates, sheep, cows, goats, pigs, chickens, dogs, cats, and rodents such as mice, rats, and guinea pigs. In preferred aspects, the subject is a human. The terms, “subject” and “patient” are used interchangeably herein.
“Administering” refers to the physical introduction of a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art.
The terms “treat,” “treating,” “treatment,” or “therapy” of a subject as used herein, refer to any type of intervention or process performed on, or administering an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, or slowing down or preventing the progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease or enhancing overall survival. Treatment can be of a subject having a disease or a subject who does not have a disease (e.g., for prophylaxis, such as vaccination).
The term “effective dose” “effective dosage,” or “effective amount” is defined as an amount of an agent sufficient to achieve or at least partially achieve a desired effect. A “therapeutically effective amount” or “therapeutically effective dosage” of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, an increase in overall survival (the length of time from either the date of diagnosis or the start of treatment for a disease that patients diagnosed with the disease are still alive), or a prevention of impairment or disability due to the disease affliction. A therapeutically effective amount or dosage of a drug includes a “prophylactically effective amount” or a “prophylactically effective dosage”, which is any amount of the drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or of suffering a recurrence of disease, inhibits the development or recurrence of the disease. The ability of a therapeutic agent to promote disease regression or inhibit the development or recurrence of the disease can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.
Various aspects of the disclosure are described in further detail in the following subsections.
Bacterial sequence-free vectors and their production are described in U.S. Pat. Nos. 9,290,778 and 9,862,954; Nafissi and Slavcev, Microbial Cell Factories 11:154 (2012); and Nafissi et al., Nucleic Acids 3(6):e165 (2014), incorporated by reference herein in their entireties. These bacterial sequence-free vectors are produced from an expression vector (e.g., a plasmid) that contains specialized “Super Sequence” (“SS” or, alternatively, “SSeq”) sites comprising target sequences for recombinases flanking each side (i.e., the 5′ and 3′ sides) of an expression cassette containing a nucleic acid sequence(s) of interest. Specifically, each SS contains a target sequence for a first recombinase, with an additional target sequence for one or more additional recombinases integrated within non-binding regions for the first recombinase. When the expression vector is present in a recombinant cell that expresses an appropriate recombinase, a bacterial sequence-free vector containing the expression cassette is separated from the backbone DNA of the expression vector. To produce a circular covalently closed (CCC) bacterial sequence-free vector, the expression vector is placed into a recombinant cell expressing a recombinase such as Cre or Flp, for example, that acts through its target sequences in the SS. To produce a linear covalently closed (LCC) bacterial sequence-free vector, also referred to herein as a ministring DNA (msDNA), the expression vector is placed into a recombinant cell expressing a recombinase such as TelN or Tel, for example, that acts through its target sequences in the SS. The bacterial sequence-free vector resulting from the recombination can then be purified from the cells and used directly as a delivery vector. See U.S. Pat. Nos. 9,290,778 and 9,862,954, Nafissi and Slavcev, and Nafissi et al.
msDNA vectors with LCC ends are torsion-free and not subject to gyrase-directed negative supercoiling during their production in E. coli. Furthermore, due to its double stranded LCC topology, integration of msDNA into a cell's chromosome causes a chromosomal break, thereby eliminating the cell from the population. Thus, msDNA eliminates any risk of insertional mutagenesis, protecting patients who are administered the msDNA from potential genotoxicity and cancer (Nafissi et. al.).
The present disclosure provides improved production of bacterial sequence-free vectors and improved bacterial sequence-free vectors. In some aspects, production of the bacterial sequence-free vectors is improved by removal of contaminating expression vector sequences. In some aspects, the bacterial sequence-free vectors is improved through its capacity for establishment in cells (i.e., transfection efficiencies), improved transgene expression (e.g., mediated by a combination of enhanced transcription and translation), and improved expansion in cells (e.g., replication and partition of the vector to daughter cells).
In some aspects, the improvements disclosed herein can be adapted to CCC or LCC vectors produced according to other methods known in the art.
Provided herein is an expression vector comprising: (a) a backbone sequence, (b) a sequence comprising: (i) an expression cassette comprising a nucleic acid sequence of interest, (ii) a first target sequence for a first recombinase flanking the 5′ side of the expression cassette, (iii) a second target sequence for the first recombinase flanking the 3′ side of the expression cassette, and (iv) one or more additional target sequences for one or more additional recombinases integrated within the first and second target sequences in non-binding regions for the first recombinase, and (c) one or more of: (i) an endonuclease target sequence integrated within the first and/or second target sequences for the first recombinase in non-binding regions for the first recombinase and the one or more additional recombinases, wherein the endonuclease target sequence is between the backbone sequence and cleavage sites for the first recombinase and the one or more additional recombinases, (ii) a synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO: 12 integrated between the 3′ end of the first target sequence for the first recombinase and the 5′ end of another enhancer or a promoter in the expression cassette, (iii) a cytomegalovirus (CMV) enhancer integrated between the 3′ end of the first target sequence for the first recombinase and the 5′ end of a promoter in the expression cassette, (iv) a 5′ untranslated region (5′UTR) comprising an intron, wherein the 5′UTR is integrated in the expression cassette between a promoter and the nucleic acid sequence of interest, (v) a vertebrate chromatin insulator integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal, (vi) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal, (vii) a scaffold/matrix attachment region (S/MAR) integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal, or (viii) a DNA nuclear targeting sequence (DTS) integrated within the first and/or second target sequences for the first recombinase in non-binding regions for the first recombinase and the one or more additional recombinases, wherein the DTS is between the expression cassette and cleavage sites for the first recombinase and the one or more additional recombinases.
A “backbone” sequence as referred to herein is the sequence of the expression vector outside of the sequence of the expression cassette and the flanking SS sites comprising the first and second target sequences of the first recombinase. The backbone sequence can include, for example, sequences for amplification and antibiotic selection of the expression vector in a host cell (e.g., E. coli) as described herein.
“Non-binding” regions for a recombinase are regions within the target sequence for the first recombinase that are not acted upon by a recombinase as described herein (e.g., not bound and/or cleaved by the recombinase).
A “cleavage site” for a recombinase is the site at which a recombinase initiates a double-strand break or single-stranded nick in the DNA associated with recombination.
In some aspects, the expression vector comprises an endonuclease target sequence integrated within the first and/or second target sequences for the first recombinase in non-binding regions for the first recombinase and the one or more additional recombinases, wherein the endonuclease target sequence is between the backbone sequence and cleavage sites for the first recombinase and the one or more additional recombinases. In some aspects, the endonuclease target sequence is integrated within the first target sequence for the first recombinase. In some aspects, the endonuclease target sequence is integrated within the second target sequence for the first recombinase. In some aspects, the endonuclease target sequence is integrated within the first and second target sequences for the first recombinase. In some aspects, the same endonuclease target sequence is integrated within the first and second target sequences for the first recombinase. In some aspects, the endonuclease target sequences integrated within the first and second target sequences for the first recombinase are for the same endonuclease. In some aspects, the endonuclease target sequence integrated within the first target sequence for the first recombinase is different from the endonuclease target sequence integrated within the second target sequence for the first recombinase. In some aspects, the endonuclease target sequence integrated within the first target sequence for the first recombinase is for a different endonuclease than the endonuclease target sequence integrated within the second target sequence for the first recombinase.
The location of the endonuclease target sequence between the backbone sequence and cleavage sites for the recombinases in the expression vector ensures that the endonuclease target sequence remains associated with the backbone sequence, and not the bacterial sequence-free vector, following recombination as described herein. Thus, following recombination, sequences containing backbone sequence and the endonuclease target site can be removed from a preparation containing bacterial sequence-free vector by exposure to an endonuclease, reducing or avoiding the need for purification steps to remove backbone sequences in methods of producing the bacterial sequence-free vector. In some aspects, the endonuclease is expressed following recombination in a host cell of a vector production system as described herein, wherein the endonuclease cuts the DNA at the endonuclease target site, and the sequence containing the backbone sequence and the endonuclease target site is degraded by an exonuclease (e.g., exonuclease V).
In some aspects, the expression vector comprises an endonuclease target sequence for a homing endonuclease. In some aspects, the endonuclease target sequence is for I-AniI, I-CeuI, I-ChuI, I-CpaI, I-CpaII, I-CreI, I-DmoI, H-DreI, I-HmuI, I-HmuII, I-LlaI, I-MsoI, PI-PfuI, PI-PkoII, I-PorI, I-PpoI, PI-PspI, I-ScaI, I-SceI, PI-SceI, I-SceII, I-SecIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-Ssp6803I, I-TevI, I-TevII, I-TevIII, PI-TliI, PI-TliII, I-Tsp061I, or I-Vdi141I. In some aspects, the endonuclease target sequence is for I-SceI. In some aspects, the endonuclease target sequence is for PI-SceI. Target sequences for homing endonucleases are well-known in the art.
In some aspects, the expression vector comprises an endonuclease target sequence for an endonuclease used in genome editing, including an endonuclease that is part of a nuclease genome editing system. In some aspects, the nuclease genome editing system is a Clustered Regularly Interspaced Short Palindromic Repeats-Cas (CRISPR-Cas) system, a Transcription Activator-Like Effector Nuclease (TALEN) system, a Zinc-Finger Nuclease (ZFN) system, or a meganuclease system.
In some aspects, the expression vector comprises an endonuclease target sequence for a Cas endonuclease. In some aspects, the Cas endonuclease is Cas9 (e.g., a Streptococcus pyogenes Cas 9 (SpCas9), a Staphylococcus aureus Cas9 (SaCas9), a Francisella novicida Cas9 (FnCas9), or a Neisseria meningitides Cas9 (NmCas9)), a Cas9 variant (e.g., Cas9β2, xCas9, SpCas9-NG, SpCas9-NRRH, SpCas9-NRCH, SpCas9-NRTH, SpG, SpRY), Cas3, Cas12 (e.g., Cas12a, Cas12b, Cas12c, Cas12d, or Cas12e), Cas13 (e.g., Cas13a, Cas13b, Cas13c, or Cas13d), or Cas14. In some aspects, an endonuclease target sequence for a Cas endonuclease as used herein is homologous to a guide RNA (gRNA) targeting sequence and includes a protospacer adjacent motif (PAM) recognized by a Cas endonuclease. Sequences homologous to gRNA targeting sequences with PAM sites can be routinely designed based on well-known CRISPR systems. The gRNA comprises a fusion of a targeting RNA (crRNA) sequence and a trans-activating RNA (tracrRNA) sequence, which interact and function to direct the Cas endonuclease to the endonuclease target site and catalyze cleavage.
In some aspects, the expression vector comprises a synthetic enhancer comprising a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 12 integrated between the 3′ end of the first target sequence for the first recombinase and the 5′ end of another enhancer or a promoter in the expression cassette. In some aspects, the expression vector comprises a synthetic enhancer comprising the nucleic acid sequence of SEQ ID NO:12 integrated between the 3′ end of the first target sequence for the first recombinase and the 5′ end of another enhancer or a promoter in the expression cassette. In some aspects, the synthetic enhancer comprises multiple contiguous copies of the nucleic acid sequence, such as, for example, 1, 2, 3, 4, 5, or more contiguous copies. In some aspects, the synthetic enhancer comprises 3 contiguous copies of the nucleic acid sequence. In some aspects, the synthetic enhancer comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:46. In some aspects, the synthetic enhancer comprises the nucleic acid sequence of SEQ ID NO:46. In some aspects, the synthetic enhancer is integrated at the 5′ end of a chicken β-actin promoter. In some aspects, a chimeric intron comprising a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:47 is integrated at the 3′ end of the chicken β-actin promoter and 5′ to the nucleic acid sequence of interest. In some aspects, a chimeric intron comprising the nucleic acid sequence of SEQ ID NO:47 is integrated at the 3′ end of the chicken β-actin promoter and 5′ to the nucleic acid sequence of interest.
In some aspects, the expression vector comprises a CMV enhancer integrated between the 3′ end of the first target sequence for the first recombinase and the 5′ end of a promoter in the expression cassette. In some aspects, the CMV enhancer is integrated at the 3′ end of a synthetic enhancer comprising a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:12. In some aspects, the CMV enhancer is integrated at the 3′ end of a synthetic enhancer comprising the nucleic acid sequence of SEQ ID NO: 12. In some aspects, the CMV enhancer is integrated at the 3′ end of multiple contiguous copies of the synthetic enhancer, such as, for example, at the 3′ end of 1, 2, 3, 4, 5, or more contiguous copies of the synthetic enhancer. In some aspects, the CMV enhancer is integrated at the 3′ end of 3 contiguous copies of the synthetic enhancer. In some aspects, the CMV enhancer is integrated at the 3′ end of a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:46. In some aspects, the CMV enhancer is integrated at the 3′ end of the nucleic acid sequence of SEQ ID NO:46. In some aspects, a CMV promoter is integrated at the 3′ end of the CMV enhancer and 5′ to the nucleic acid sequence of interest.
In some aspects, the expression vector comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39 integrated between the first target sequence for the first recombinase and the nucleic acid sequence of interest. In some aspects, the expression vector comprises the nucleic acid sequence of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39 integrated between the first target sequence for the first recombinase and the nucleic acid sequence of interest. In some aspects, a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39, or the nucleic acid sequence of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39, comprises all regulatory elements in the expression cassette located 5′ to the nucleic acid sequence of interest.
In some aspects, the expression vector comprises a 5′UTR comprising an intron, wherein the 5′UTR (i.e., the 5′UTR comprising the intron) is integrated in the expression cassette between a promoter and the nucleic acid sequence of interest.
In some aspects, the 5′UTR is for improving transgene transcript splicing and translation from the expression vector or from a bacterial sequence-free vector produced from the expression vector as compared to the same expression vector or bacterial sequence-free vector, respectively, lacking the 5′UTR.
In some aspects, the intron comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:1. In some aspects, the intron comprises the nucleic acid sequence of SEQ ID NO:1.
In some aspects, the 5′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:2, which is an optimized 5′UTR with an internal minimal intron, also referred to herein as “5′UTR1.” In some aspects, the 5′UTR comprises the nucleic acid sequence of SEQ ID NO:2.
In some aspects, the 5′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:4. In some aspects, the 5′UTR comprises the nucleic acid sequence of SEQ ID NO:4.
In some aspects, the 5′UTR further comprises a non-coding sequence integrated within the intron.
In some aspects, the intron is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:1, or comprises SEQ ID NO: 1, and the non-coding sequence is integrated between two of the nucleotides in the intron corresponding to any two nucleotides from positions 25 to 55 of SEQ ID NO:1.
In some aspects, the non-coding sequence is non-prokaryotic and non-viral. In some aspects, the non-coding sequence is a eukaryotic sequence. In some aspects, the non-coding sequence comprises an intron, a ubiquitous chromatin opening element (UCOE), an S/MAR, an SV40 enhancer sequence (e.g., one or more than one SV40 enhancer sequences, such as two, three, four, five or more SV40 enhancer sequences), a vertebrate chromatin insulator (e.g., cHS4), a WPRE, or any combination thereof.
In some aspects, the non-coding sequence comprises an S/MAR. In some aspects, the S/MAR is MAR-5, provided herein as SEQ ID NO:9.
In some aspects, the 5′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:3. In some aspects, the 5′UTR comprises SEQ ID NO:3.
In some aspects, the 5′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:5. In some aspects, the 5′UTR comprises SEQ ID NO:5.
In some aspects, the 5′UTR is integrated in the expression cassette between a chicken β-actin promoter and the nucleic acid sequence of interest.
In some aspects, the 5′UTR is integrated in the expression cassette between a CMV promoter and the nucleic acid sequence of interest.
In some aspects, the 5′UTR is integrated in the expression cassette between a promoter and the nucleic acid sequence of interest, wherein the promoter is integrated at the 3′ end of a CMV enhancer. In some aspects, the CMV enhancer is integrated at the 3′ end of a synthetic enhancer comprising a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:12. In some aspects, the CMV enhancer is integrated at the 3′ end of a synthetic enhancer comprising the nucleic acid sequence of SEQ ID NO: 12. In some aspects, the CMV enhancer is integrated at the 3′ end of multiple contiguous copies of the synthetic enhancer, such as, for example, at the 3′ end of 1, 2, 3, 4, 5, or more contiguous copies of the synthetic enhancer. In some aspects, the CMV enhancer is integrated at the 3′ end of 3 contiguous copies of the synthetic enhancer. In some aspects, the CMV enhancer is integrated at the 3′ end of a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:46. In some aspects, the CMV enhancer is integrated at the 3′ end of a nucleic acid sequence of SEQ ID NO:46.
In some aspects, the expression vector comprises a polyadenylation signal integrated at the 3′ end of the nucleic acid sequence of interest. In some aspects, the polyadenylation signal comprises a Xenopus laevis beta-globin polyadenylation signal, a human beta-globin polyadenylation signal, or a hybrid Xenopus laevis and human beta-globin polyadenylation signal. In some aspects, the polyadenylation signal comprises multiple copies of a Xenopus laevis beta-globin polyadenylation signal, a human beta-globin polyadenylation signal, or a hybrid Xenopus laevis and human beta-globin polyadenylation signal, such as, for example, 1, 2, 3, 4, or 5 copies. In some aspects, the polyadenylation signal comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO:15. In some aspects, the polyadenylation signal comprises the nucleic acid sequence of SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15. In some aspects, a polyadenylic acid tail (i.e., poly(A) tail is located at the 3′ end of the polyadenylation signal. In some aspects, the poly(A) tail is 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, or more residues in length. In some aspects, the sequence comprising the polyadenylation signal and the poly(A) tail is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. In some aspects, the sequence comprising the polyadenylation signal and the poly(A) tail comprises SEQ ID NO: 16, SEQ ID NO:17, or SEQ ID NO:18.
In some aspects, the expression vector comprises a vertebrate chromatin insulator in the expression cassette. In some aspects, the vertebrate chromatin insulator is 5′-HS4 chicken-β-globin insulator (cHS4). See, e.g., Benabdellah et al., PLOS ONE 9(1): e84268 (2014); Lu et al., FEBS Open Bio 10: 644-656 (2020); Hanawa et al., Mol. Ther. 17(4): 667-674 (2009); Walters et al., Mol. Cell. Biol. 19(5): 3714-3726 (1999). In some aspects, the vertebrate chromatin insulator is integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal as described herein. In some aspects, the vertebrate chromatin insulator is integrated within the intron of a 5′UTR as described herein.
In some aspects, the vertebrate chromatin insulator comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:8. In some aspects, the vertebrate chromatin insulator comprises SEQ ID NO:8.
In some aspects, the vertebrate chromatin insulator is for improving establishment (i.e., transfection efficiency) of the expression vector or a bacterial sequence-free vector produced from the expression vector as compared to the same expression vector or bacterial sequence-free vector, respectively, without the vertebrate chromatin insulator.
In some aspects, the expression vector comprises a WPRE in the expression cassette. See, e.g., Higashimoto et al., Gene Therapy 14: 1298-1304 (2007). In some aspects, the WPRE is integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal as described herein. In some aspects, the WPRE is integrated in the expression cassette at the 3′ end of a S/MAR as described herein and the 5′ end of a polyadenylation signal as described herein. In some aspects, the WPRE is integrated within the intron of a 5′UTR as described herein.
In some aspects, the WPRE comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:11. In some aspects, the WPRE comprises SEQ ID NO:11.
In some aspects, the WPRE improves expression of the transgene from the expression vector or the bacterial sequence-free vector produced from the expression vector as compared to the same expression vector or bacterial sequence-free vector, respectively, lacking the WPRE.
In some aspects, the expression vector comprises a S/MAR in the expression cassette. See, e.g., Martens et al., Mol. Cell. Biol. 22(8): 2598-2606 (2002); Narwade et al., Nucleic Acids Res. 47(14): 7247-7261 (2019). In some aspects, the S/MAR is integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal. In some aspects, the S/MAR is integrated in the expression cassette at the 3′ end of a nucleic acid sequence of interest and the 5′ end of a WPRE as described herein. In some aspects, the S/MAR is integrated within the intron of a 5′UTR as described herein.
In some aspects, the S/MAR is MAR-3, MAR-4, or MAR-5, which are fragments of human beta-interferon MAR. See, e.g., Wang et al., Mol. Biol. Cell 30: 2761-2770 (2019). In some aspects, the S/MAR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:9. In some aspects, the S/MAR comprises SEQ ID NO:9.
In some aspects, the S/MAR is human cytotoxic serine protease-B (CSP-B) MAR or CSP-C MAR. See, e.g., Hanson and Ley, Blood 79(3): 610-618 (1992); Klein et al., Tissue Antigens 35(5):220-228 (1990). In some aspects, the S/MAR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:10. In some aspects, the S/MAR comprises SEQ ID NO:10.
In some aspects, the S/MAR is for improving expression levels, stability, and/or durability (e.g., by episomal maintenance and replication, such as expansion and partition of the vector to daughter cells, and/or by preventing epigenetic silencing) of the expression vector or a bacterial sequence-free vector (produced from the expression vector as compared to the same expression vector or bacterial sequence-free vector, respectively, lacking the S/MAR.
In some aspects, the expression vector comprising any one of more of (c)(i)-(c)(vii) as described above (i.e., without a DTS) further comprises an enhancer sequence flanking each side of the first and second target sequences for the first recombinase. In some aspects, the enhancer sequence flanking each side of the first and second target sequences for the first recombinase is at least two enhancer sequences flanking each side of the first and second target sequences for the first recombinase. In some aspects, the enhancer sequence is a SV40 enhancer sequence.
In some aspects, the expression vector comprises a DTS. In some aspects, the DTS is integrated within the first and/or second target sequences for the first recombinase in non-binding regions for the first recombinase and the one or more additional recombinases, wherein the DTS is between the expression cassette and cleavage sites for the first recombinase and the one or more additional recombinases. In some aspects, the DTS is a SV40 enhancer sequence. In some aspects, the DTS is cell-specific. In some aspects, the DTS is specific for smooth muscle cells, embryonic stem cells, type II pneumonocytes, endothelial cells, or osteoblasts.
The location of the DTS between the expression cassette and cleavage sites for the recombinases in the expression vector ensures that the DTS remains associated with the bacterial sequence-free vector, and not the backbone sequence, following recombination as described herein.
In some aspects, the expression vector comprises a UCOE in the expression cassette. See, e.g., Müller-Kuller et al., Nucleic Acids Res. 43(3): 1577-1592 (2015); Skipper et al., BMC Biotechnol. 19:75 (2019); Rudina et al., bioRxiv, doi.org/10.1101/626713 (2019); Neville et al., Biotechnol. Adv. 35(5): 557-564 (2017). In some aspects, the UCOE is located between the 3′ end of the first target sequence for the first recombinase and the 5′ end of a promoter or any enhancer in the expression cassette. In some aspects, the UCOE is integrated within the intron of a 5′UTR as described herein.
In some aspects, the UCOE is A2UCOE. In some aspects, the UCOE comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:6. In some aspects, the UCOE is SEQ ID NO:6.
In some aspects, the UCOE is SRF-UCOE. See, e.g., International Publication No. WO2020223160. In some aspects, the UCOE comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:7. In some aspects, the UCOE is SEQ ID NO:7.
In some aspects, the UCOE improves expression of the transgene from the expression vector or a bacterial sequence-free vector produced from the expression vector as compared to the same expression vector or bacterial sequence-free vector, respectively, lacking the UCOE.
In some aspects, the expression vector comprises Enhancer-1 in the expression cassette. In some aspects, Enhancer-1 is integrated between the 3′ end of the first target sequence for the first recombinase and the 5′ end of a promoter or any other enhancer in the expression cassette. In some aspects, Enhancer-1 is integrated between the 3′ end of a UCOE and the 5′ end of a CMV enhancer. In some aspects, Enhancer-1 comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 12. In some aspects, Enhancer-1 is SEQ ID NO: 12.
In some aspects, the expression vector comprises a CMV, EF1, SV40, CAG, Rho, VDM2, HCR, or HLP promoter, or variant thereof, in the expression cassette. In some aspects, the expression vector comprises a CMV promoter variant in the expression cassette. See, e.g., International Publication No. WO2012099540; Xu et al., Bioengineered 10(1): 548-560, DOI: 10.1080/21655979.2019.1684863 (2019).
In some aspects, the expression vector comprises an EF1-alpha promoter in the expression cassette. In some aspects, the expression vector comprises a CMV enhancer and an EF1-alpha promoter in the expression cassette.
In some aspects, the expression vector comprises a 3′UTR in the expression cassette comprising two copies of a beta-globin polyadenylation signal. In some aspects, the 3′UTR is integrated between the nucleic acid sequence of interest and the 5′ end of the second target sequence for the first recombinase.
In some aspects, the 3′UTR comprises two copies of a Xenopus laevis beta-globin polyadenylation signal. In some aspects, the 3′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:13. In some aspects, the 3′UTR is SEQ ID NO:13.
In some aspects, the 3′UTR comprises two copies of a human beta-globin polyadenylation signal. In some aspects, the 3′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:14. In some aspects, the 3′UTR is SEQ ID NO:14.
In some aspects, the 3′UTR comprises one copy of a Xenopus laevis beta-globin polyadenylation signal and one copy of a human beta-globin polyadenylation signal. In some aspects, the 3′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:15. In some aspects, the 3′UTR is SEQ ID NO:15.
In some aspects, the 3′UTR further comprises a poly(A) tail (i.e., at the 3′ end of the 3′UTR) comprising 100 to 120 adenine nucleotides, i.e., 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 adenine nucleotides.
In some aspects, the 3′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:16. In some aspects, the 3′UTR is SEQ ID NO:16.
In some aspects, the 3′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:17. In some aspects, the 3′UTR is SEQ ID NO:17.
In some aspects, the 3′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:18. In some aspects, the 3′UTR is SEQ ID NO:18.
The expression vector can contain any combination of the above modifications to the first and/or second target sequences and/or the expression cassette as described herein. In some aspects, the combination provides a synergistic effect.
In some aspects, the first and second target sequences for the first recombinase and the one or more additional target sequences for the one or more additional recombinases are selected from the group consisting of the PY54 pal site, the N15 telRL site, the loxP site, φK02 telRL site, the FRT site, the phiC31 attP site, and the λ attP site. In some aspects, the expression vector comprises each of the target sequences. In some aspects, the expression vector comprises the pal site and the telRL, loxP, and FRT recombinase target binding sequences integrated within the pal site. In some aspects, the first and second target sequences for the first recombinase each comprise the nucleic acid sequence of SEQ ID NO:33.
In some aspects, the nucleic acid sequence of interest in any of the expression cassettes described herein comprises a sequence encoding: a polypeptide, an RNA (messenger RNA (mRNA), micro-RNA (miRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), ribozyme, or antisense RNA), or a non-coding DNA (e.g., an antisense oligonucleotide). In some aspects, the nucleic acid sequence of interest is a genomic DNA sequence comprising introns and/or exons. In some aspects, the nucleic acid sequence of interest comprises a sequence encoding: an anti-cancer agent, a tumor suppressor, an apoptotic agent, an anti-angiogenesis agent, an enzyme, a cytotoxic agent, a suicide gene, a cytokine, an interferon, an interleukin, an immunomodulatory agent, an immunostimulatory agent, an immunoinhibitory agent, a chemokine, an antigen for stimulating an antigen-presenting cell, an antibody (e.g., a heavy chain and/or a light chain of an antibody, such as a monoclonal, chimeric, humanized, or human antibody, or an antigen-binding fragment thereof), a genome editing system or a portion thereof (e.g., CRISPR-Cas, TALEN, ZFN, or meganuclease systems or portions thereof, such as a Cas endonuclease or a gRNA), or an immunogenic agent (e.g., as a VLP and/or vaccine). In some aspects, the nucleic acid sequence of interest comprises sequences encoding polypeptides that are capable of forming a VLP when the nucleic acid sequence is expressed intracellularly.
Exemplary therapeutic targets and indications include: a gene associated with a monogenic disorder, including, for example, a liver, blood, or eye disorder, galactosidase alpha (GLA, e.g., for treating Fabry disease), sodium voltage-gated channel alpha subunit 1 (SCNIA, e.g., for treating dravet syndrome), ATP binding cassette subfamily A member 4 (ABCA4, e.g., for treating Stargardt disease), surfactant protein B (SP-B, e.g., for treating surfactant dysfunction disorder), surfactant protein C (SP-C, e.g., for treating surfactant dysfunction disorder), ATP-binding cassette sub-family A member 3 (ABCA3, e.g., for treating surfactant dysfunction disorder), solute carrier family 34 member 2 (SLC34A2, e.g., for treating pulmonary alveolar microlithiasis and/or testicular microlithiasis), cystic fibrosis transmembrane conductance regulator (CFTR, e.g., for treating cystic fibrosis), glutamate decarboxylase (GAD, e.g., GAD65 or GAD67, e.g., for treating Parkinson's disease), aspartoacylase gene (ASPA, also known as aminoacylase (AAC), e.g., for treating Canavan disease), aromatic L-amino acid decarboxylase (AADC, e.g., for treating Parkinson's disease and/or for treating AADC deficiency), neurturin (NRTN, e.g., for treating Parkinson's disease), glial cell line-derived neurotrophic factor (GDNF, e.g., for treating Parkinson's disease), nerve growth factor (NGF, e.g., for treating Alzheimer's disease), tripeptidyl peptidase I (TPP1, also known as ceroid lipofuscinosis neuronal-2 (CLN2), e.g., for treating Batten disease, e.g., CLN2 disease), arylsulfatase A (ARSA, e.g., for treating metachromatic leukodystrophy), N-sulphoglucosamine sulphohydrolase (SGSH, e.g., for treating Sanfilippo syndrome, Type A), Sulfatase-modifying factor 1 (SUMF1, e.g., for treating Sanfilippo syndrome, Type A), N-acetyl-alpha-glucosaminidase (NAGLU, e.g., for treating Sanfilippo syndrome, Type B), survival of motor neuron 1 (SMN1, e.g., for treating spinal muscular atrophy 1), retinal pigment epithelium-specific 65 kDa protein (RPE65, also known as retinoid isomerohydrolase, e.g., for treating Leber's congenital amaurosis), Rab escort protein 1 (REP1, e.g., for treating choroideremia), retinoschisin 1 (RS1, e.g., for treating X-linked juvenile retinoschisis), alpha-1 antitrypsin (AAT, e.g., for treating hereditary emphysema or AAT deficiency), minidystrophin (e.g., for treating Duchenne's muscular dystrophy), α-sarcoglycan (αSG, e.g., for treating Duchenne's muscular dystrophy or limb girdle muscular dystrophy type 2), β-sarcoglycan (BSG), γ-sarcoglycan (γSG, e.g., for treating limb girdle muscular dystrophy type 2), δ-sarcoglycan (γSG), ipoprotein lipase (LPL, e.g., for treating familial LPL deficiency), acid alpha-glucosidase (GAA, e.g., for treating Pompe disease), tumor necrosis factor receptor:Fc (TNFR:Fc, e.g., for treating arthritis, e.g., inflammatory arthritis), sarcoplasmic/endoplasmic reticulum Ca(2+)ATPase 2a (SERCA2a, e.g., for treating congestive heart failure), Factor VIII or Factor IX (FVIII or FIX, e.g., for treating hemophilia B), porphobilinogen deaminase gene (PBGD, e.g., for treating acute intermittent porphyria), soluble fms-like tyrosine kinase-1 (sFLT1, e.g., for treating age-related macular degeneration or cancer, e.g., ovarian cancer), a soluble chimeric vascular endothelial growth factor (VEGF) receptor comprising domains of VEGFR-1 and VEGF-R2 (e.g., for treating cancer, e.g., melanoma or colon cancer), soluble VEGFR3 (e.g., for treating cancer, e.g., endometrial cancer), a soluble VEGF-C decoy receptor (sVEGFR3-Fc, e.g., for treating cancer, e.g., melanoma, renal cell carcinoma, or prostate cancer), pigment epithelium-derived growth factor (PEDF, e.g., for treating cancer, e.g., Lewis lung carcinoma), a neutralizing monoclonal antibody against VEGFR2 (e.g., DC101, e.g., for treating cancer, e.g., melanoma or glioblastoma), endostatin (e.g., for treating cancer, e.g., bladder or pancreatic cancer), angiostatin (e.g., for treating cancer, e.g., liver cancer), both endostatin and angiostatin (i.e., as a bicistronic sequence, e.g., for treating cancer, e.g., ovarian or prostate cancer), an endostatin mutant (i.e., P1254A-endostatin, e.g., for treating cancer, e.g., ovarian cancer), antiangiogenic domain of TSP-1 (3TSR, e.g., for treating cancer, e.g., pancreatic cancer), tissue factor pathway inhibitor-2 (TFPI-2, e.g., for treating cancer, e.g., glioblastoma), a fragment of plasminogen (e.g., kringle 5, e.g., for treating cancer, e.g., ovarian cancer), plasminogen kringle 1-5 (e.g., for treating cancer, e.g., melanoma or lung cancer), siRNA against an unfolded protein response protein (UPR; e.g., IRE1α, XBP-1, or ATF6, e.g., for treating cancer, e.g., breast cancer), vasostatin (e.g., for treating cancer, e.g., lung cancer), herpes simplex virus type 1 thymidine kinase (HSV-TK, e.g., for treating cancer, e.g., breast cancer), sc39TK (e.g., for treating cancer, e.g., cervical cancer), diphtheria toxin A (DTA, e.g., for treating cancer, e.g., cervical cancer or myeloma), p53 upregulated modulator of apoptosis (PUMA, e.g., for treating cancer, e.g., cervical cancer or myeloma), tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL, e.g., for treating cancer, e.g., lymphoma, hepatocellular carcinoma, head and neck squamous cell carcinoma (i.e., head and neck cancer), or glioblastoma), soluble TRAIL (e.g., for treating cancer, e.g., liver cancer or lung adenocarcinoma), IFN-β (e.g., for treating cancer, e.g., colorectal cancer, lung cancer, neuroblastoma, or glioblastoma multiforme), IFN-α (e.g., for treating cancer, e.g., metastatic melanoma), a CD-40 ligand (CD40L) or CD40L mutant (e.g., for treating cancer, e.g., lung cancer), melanoma differentiation-associated gene-7 and interleukin 24 (mda-7 and IL24, e.g., for treating cancer, e.g., Ehrlich ascites tumor), apoptotin and IL24 (e.g., for treating cancer, e.g., liver cancer), IL24 (e.g., for treating cancer, e.g., mixed-lineage leukemia (MLL)/AF4 positive acute lymphoblastic leukemia (ALL)), IL15 (e.g., for treating cancer, e.g., metastatic hepatocellular carcinoma), secondary lymphoid tissue chemokine (SLC, e.g., for treating cancer, e.g., liver cancer), Nk4 (the N-terminal hairpin and subsequent four kringle domains of hepatocyte growth factor (HGF), e.g., for treating cancer, e.g., metastatic Lewis lung carcinoma), tumor necrosis factor superfamily member 14 (TNFSF14, also known as LIGHT, e.g., for treating cancer, e.g., cervical cancer), Granulocyte-macrophage colony-stimulating factor (GM-CSF, e.g., for treating cancer), TNF-α (e.g., for treating cancer, e.g., glioma), a dominant negative mutant of survivin (e.g., C84A or T34A, e.g., for treating cancer, e.g., colon or gastric cancer), the C-terminal fragment of the human telomerase reverse transcriptase (hTERTC27, e.g., for treating cancer, e.g., glioblastoma multiforme), maspin (e.g., for treating cancer, e.g., prostate cancer), nm23H1 (e.g., for treating cancer, e.g., metastatic ovarian cancer), kringle 1 domain of human hepatocyte growth factor (HGFK1, e.g., for treating cancer, e.g., colorectal carcinoma), anti-calcitonin ribozyme (e.g., for treating cancer, e.g., prostate cancer), eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1, e.g., for treating cancer, e.g., lung cancer), C-X-C motif chemokine receptor 2 (CXCR2) C-tail sequence (e.g., for treating cancer, e.g., pancreatic cancer), alpha-tocopherol-associated protein (TAP, e.g., for treating cancer, e.g., prostate cancer), trichosanthin (e.g., for treating cancer, e.g., hepatocellular carcinoma), decorin (e.g., for treating cancer, e.g., glioblastoma multiforme), cathelicidin (e.g., for treating cancer, e.g., colon cancer), Niemann-Pcik type C2 (NPC2, e.g., for treating cancer, e.g., hepatocellular carcinoma), Mullerian inhibiting substance (MIS, e.g., for treating cancer, e.g., ovarian cancer), p53 (e.g., for treating cancer, e.g., bronchoalveolar cancer), shRNA against highly expressed in cancer 1 (Hec1, e.g., for treating cancer, e.g., glioma), shRNA against Epstein-Barr virus latent membrane protein-1 (EBV LMP-1, e.g., for treating cancer, e.g., nasopharyngeal cancer), anti-sense RNA against human papilloma virus 16 E7 oncogene (HPV16-E7, e.g., for treating cancer, e.g., cervical cancer), shRNA against androgen receptor (AR, e.g., for treating cancer, e.g., prostate cancer), siRNA against Snail (also known as SNA1, e.g., for treating cancer, e.g., pancreatic cancer), siRNA against Slug (i.e., the protein product of SNAI2, e.g., for treating cancer, e.g., cholangiocarcinoma (liver cancer)), shRNA against Four and a half LIM-only protein 2 (FHL2, e.g., for treating cancer, e.g., colon cancer), miR-26a (e.g., for treating cancer, e.g., hepatocellular carcinoma), HPV 16 structural protein L1 (HPV16-L1, e.g., for treating cancer, e.g., cervical cancer), HPV 16 E5, E6, and E7 oncogenes (HPV16 E5/E6/E7, e.g., for treating cancer, e.g., cervical cancer), B-cell leukemia/lymphoma 1 (BLC1) idiotype (e.g., for treating cancer, e.g., B cell leukemia/lymphoma 1), EBV LMP1 and LMP2 fused to heat shock protein (EBV LMP2/1-hsp, e.g., for treating cancer, e.g., nasopharyngeal carcinoma), carcinoembryonic antigen (CEA, e.g., for treating cancer, e.g., colon cancer), soluble form of B and T lymphocyte attenuator in combination with a heat shock protein (BTLA and HSP70, e.g., for treating cancer, e.g., melanoma pulmonary metastasis), HPV16-L1/E7 (e.g., for treating cancer, e.g., cervical cancer), HPV16-L1 (e.g., for treating cancer, e.g., cervical cancer), an anti-EGFR antibody (e.g., 14D1, e.g., for treating cancer, e.g., vulvar carcinoma), an anti-death receptor 5 (DR5) antibody (e.g., adximab, e.g., for treating cancer, e.g., liver or colon cancer), an anti-Enolase 1 (ENOI1) antibody (e.g., for treating cancer, e.g., pancreatic ductal adenocarcinoma), an anti-VEGFA antibody (e.g., bevacizumab, e.g., for treating cancer, e.g., metastatic lung cancer or ovarian cancer), the Mucin 1 (MUC1) antigen (e.g., for treating cancer, e.g., gastric cancer), or an aquaporin (e.g., hAQP1, e.g., for treating irradiation induced parotid salivary hypofunction, i.e., xerostomia).
In some aspects, the nucleic acid sequence of interest is for use in gene editing (e.g., gene therapy, including treatment of a genetic deficiency, disorder, or disease).
In some aspects, the nucleic acid sequence of interest is for insertion into a target site for gene editing (i.e., a site within a DNA or RNA sequence that is the target of gene editing). A target site for gene editing includes any genetic element, such as any cis element. In some aspects, the target site for gene editing is located within an exon of a gene, an intron of a gene, or a regulatory element of a gene.
In some aspects, the gene editing comprises an endonuclease. In some aspects, the endonuclease is associated with a genome editing system. In some aspects, the endonuclease is, for example, a homing endonuclease, a site-specific nuclease, a structure-guided nuclease, or an RNA-guided nuclease (e.g., a transposon-encoded RNA-guided nuclease).
In some aspects, the gene editing comprises a genome editing system that produces a double-strand break within the target site for gene editing. In some aspects, the genome editing system is a CRISPR-Cas, TALEN, ZFN, or meganuclease gene editing system.
In some aspects, the nucleic acid sequence of interest is inserted into the target site for gene editing by non-homologous end joining at the double-strand break. In some aspects, the double-strand break is produced by a CRISPR-Cas system. In some aspects, an expression vector as described herein comprises a Cas endonuclease target sequence (i.e., a sequence homologous to a gRNA targeting sequence) located between the first and second target sequences for the first recombinase and the nucleic acid sequence of interest (i.e., between the 5′ Super Sequence and the nucleic acid sequence of interest and between the 3′ Super Sequence and the nucleic acid sequence of interest), wherein the target site for gene editing (e.g., a target site in a chromosome) comprises the same Cas endonuclease target sequence. For example, processing of the Cas endonuclease target sequences flanking the nucleic acid sequence in a bacterial sequence-free vector (e.g., msDNA) produced from the expression vector results in removal of the Super Sequences, rendering a linear covalently closed bacterial sequence-free vector such as msDNA to instead be linear and open-ended, with reactive ends that are amenable to non-homologous end-joining events.
In some aspects, the nucleic acid sequence of interest is inserted into the target site for gene editing by homology-directed repair, which occurs through recombination between sequences flanking the double-strand break and homologous sequences associated with the nucleic acid sequence of interest.
In some aspects, the nucleic acid sequence of interest has sufficient homology with sequences flanking the double-strand break to support homology-directed repair.
In some aspects, the nucleic acid sequence of interest is flanked by 5′ and 3′ homology arms (i.e., sequences that have sufficient homology with sequences flanking the double-strand break to mediate homology-directed repair).
In some aspects, sufficient homology to mediate homology-directed repair comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% homology between the nucleic acid sequence of interest and the sequences flanking the double-strand break or between homology arms flanking the nucleic acid sequence of interest and the sequences flanking the double-strand break. In some aspects, a sequence flanking the double-strand break is within about 100 bases, about 90 bases, about 80 bases, about 70 bases, about 60 bases, about 50 bases, about 45 bases, about 40 bases, about 35 bases, about 30 bases, about 25 bases, about 20 bases, about 15 bases, about 10 bases, or about 5 bases of the double-strand break, or immediately flanks the double-strand break.
In some aspects, the homology-directed repair is by a CRISPR-Cas system. In some aspects, an expression vector as described herein comprises the CRISPR-Cas system. In some aspects, the expression vector comprises a tRNA-gRNA polycistron flanking each side of a sequence encoding a Cas endonuclease (e.g., an immunosilenced Cas9-B2). An exemplary aspect is shown in
In some aspects, the nucleic acid sequence of interest is homologous to the target site for gene editing and comprises one or more nucleotide insertions, deletions, inversions, or rearrangements as compared to the target site. In some aspects, the nucleic acid of interest is a genomic sequence, a coding region, an exon, an intron, or any portion thereof that replaces a homologous sequence at the target site.
In some aspects, the nucleic acid sequence of interest is non-homologous to the target site for gene editing.
In some aspects, the nucleic acid sequence of interest restores a missing function, corrects an abnormal function, or provides an additional function associated with the target site for gene editing.
In some aspects, the nucleic acid sequence of interest is for knockout of gene expression associated with a target site for gene editing (i.e., gene silencing).
In some aspects, the nucleic acid sequence of interest is for in vivo gene editing.
In some aspects, the nucleic acid sequence of interest is for in vitro gene editing.
In some aspects, the nucleic acid sequence of interest is for ex vivo gene editing (e.g., cell therapy, such as chimeric antigen receptor (CAR) T cell therapy).
In some aspects, the gene editing comprises an epigenetic modification, and an expression vector as described herein comprises an epigenetic effector molecule as the nucleic acid of interest. In some aspects, the epigenetic effector molecule mediates, for example, acetylation or deacetylation, methylation or demethylation, or phosphorylation or dephosphorylation. In some aspects, the epigenetic effector molecule inhibits acetylation or deacetylation, methylation or demethylation, or phosphorylation or dephosphorylation. In some aspects, the epigenetic modification is a histone modification. In some aspects, the histone modification is histone acetylation and the nucleic acid of interest is a histone acetyltransferase. In some aspects, the histone modification is histone deacetylation and the nucleic acid of interest is a histone deacetylase. In some aspects, the epigenetic modification is a DNA modification. In some aspects, the DNA modification is DNA methylation and the nucleic acid of interest is a DNA methylase. In some aspects, the DNA modification is DNA demethylation and the nucleic acid of interest is a DNA demethylase. In some aspects, the epigenetic effector molecule is fused to a targeting molecule, such as a DNA-binding molecule to target the effector to a location on the chromosome.
In some aspects, the expression cassette is polygenic, i.e., the expression cassette comprises two or more nucleic acid sequences of interest encoding two or more polypeptides, respectively.
In some aspects, the expression cassette comprises a single open reading frame comprising a nucleic acid sequence encoding a self-cleaving peptide between each nucleic acid sequence encoding a polypeptide, such that the translation product of the expression cassette is cleaved intracellularly into two or more polypeptides. In some aspects, the self-cleaving peptide is a 2A self-cleaving peptide. In some aspects, the 2A self-cleaving peptide is P2A from porcine teschovirus-1. In some aspects, the 2A self-cleaving peptide is T2A from thosea asigna virus 2A. In some aspects, the self-cleaving peptide comprises any one or more of 2A, P2A, and T2A. In some aspects, the self-cleaving peptide comprises P2A and T2A.
In some aspects, the expression cassette further comprises a nucleic acid sequence encoding a marker for gene expression. In some aspects, the marker for gene expression is a fluorescent reporter gene, such as green fluorescent protein (GFP, e.g., enhanced GFP (eGFP)), red fluorescent protein (RFP), yellow fluorescent protein (YFP), or near-infrared fluorescent protein (iRFP); a bioluminescent reporter genes such as luciferase (e.g., nanoluciferase, i.e., NanoLuc® (NLuc), England et al., Bioconjug. Chem. 27(5):1175-1187 (2016), Promega Corporation); a selectable antibiotic marker; or LacZ. In some aspects, the expression cassette comprises a nucleic acid sequence encoding a self-cleaving peptide between the nucleic acid sequence encoding a marker for gene expression and any other nucleic acid sequence encoding a polypeptide.
The expression cassette can contain any expression control region known to those of skill in the art operably linked to the nucleic acid sequence(s) of interest. In some aspects, the expression control region is a promoter, enhancer, operator, repressor, ribosome binding site, translation leader sequence, intron, polyadenylation recognition sequence, RNA processing site, effector binding site, stem-loop structure, transcription termination signal, or a combination thereof.
In some aspects, the expression vector is for producing a bacterial sequence-free vector. In some aspects, the bacterial sequence-free vector is a circular covalently closed vector. In some aspects, the bacterial sequence-free vector is a linear covalently closed vector.
Provided herein is a vector production system comprising recombinant cells encoding a recombinase under the control of an inducible promoter, wherein the recombinant cells comprise an expression vector as described herein that contains first and second target sequences for a first recombinase and one or more additional target sequences for one or more additional recombinases, and wherein the recombinase targets the first and second target sequences for the first recombinase or one of the one or more additional target sequences for the one or more additional recombinases.
Suitable host cells for use in the vector production system include microbial cells, for example, bacterial cells such as E. coli cells, and yeast cells such as S. cerevisiae. Mammalian host cells can also be used, including Chinese hamster ovary (CHO) cells (e.g., the K1 lineage (ATCC CCL 61) or the Pro5 variant (ATCC CRL 1281)); fibroblast-like cells derived from SV40-transformed African Green monkey kidney of the CV-1 lineage (ATCC CCL 70), of the COS-1 lineage (ATCC CRL 1650), or of the COS-7 lineage (ATCC CRL 1651; murine L-cells; murine 3T3 cells (ATCC CRL 1658); murine C127 cells; human embryonic kidney cells of the 293 lineage (ATCC CRL 1573); human carcinoma cells including those of the HeLa lineage (ATCC CCL 2); and neuroblastoma cells of the lines IMR-32 (ATCC CCL 127), SK-N-MC (ATCC HTB 10), or SK-N-SH (ATCC HTB 11).
Suitable recombinases catalyze DNA exchange at a target sequence for a recombinase as described herein including, but not limited to, TelN, Tel, Tel (gp26 K02 phage), Cre, Flp, phiC31, Int, and other lambdoid phage integrases, e.g. phi 80, HK022 and HP1 recombinases. In some aspects, the recombinase is TelN, Tel, Cre, or Flp.
In some aspects, the recombinant cells further encode an endonuclease under the control of an inducible promoter, wherein the endonuclease targets an endonuclease target sequence in the expression vector.
Suitable endonucleases cleave polynucleotides at the endonuclease target sequence. In some aspects, the endonuclease is a homing endonuclease. In some aspects, the homing endonuclease is I-AniI, I-CeuI, I-ChuI, I-CpaI, I-CpaII, I-CreI, I-DmoI, H-DreI, I-HmuI, I-HmuII, I-LlaI, I-MsoI, PI-PfuI, PI-PkoII, I-PorI, I-PpoI, PI-PspI, I-ScaI, I-SceI, PI-SceI, I-SceII, I-SecIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-Ssp6803I, I-TevI, I-TevII, I-TevIII, PI-TliI, PI-TliII, I-Tsp061I, or I-Vdi141I. In some aspects, the endonuclease is I-SceI. In some aspects, the endonuclease is PI-SceI. In some aspects, the recombinant cells encode a nuclease genome editing system comprising the endonuclease. In some aspects, the genome editing system is a CRISPR-Cas, a TALEN, a ZFN, or a meganuclease system. In some aspects, the nuclease genome editing system is a Class 1 or a Class 2 CRISPR-Cas system. In some aspects, the nuclease genome editing system is Type I, II, III, IV, V, or VI CRISPR-Cas system. In some aspects, the Cas endonuclease in the CRISPR-Cas system is Cas9 (e.g., a SpCas9, a SaCas9, a FnCas9, or a NmCas9), a Cas9 variant (e.g., CasB9, xCas9, SpCas9-NG, SpCas9-NRRH, SpCas9-NRCH, SpCas9-NRTH, SpG, SpRY), Cas3, Cas12 (e.g., Cas12a, Cas12b, Cas12c, Cas12d, or Cas12e), Cas13 (e.g., Cas13a, Cas13b, Cas13c, or Cas13d), or Cas14.
Recombinant host cells encoding a recombinase, or a recombinase and an endonuclease, are prepared using well-known techniques. For example, a nucleic acid sequence encoding a selected recombinase or endonuclease is introduced into the cell using a suitable vector under appropriate conditions for cell transformation. The recombinant host cells can be transformed via an expression vector, or by integration of a recombinase-encoding and/or endonuclease-encoding nucleic acid sequence into the host cell genome. In aspects where the endonuclease is associated with a nuclease genome editing system, the host cell can be designed to encode all of the components of the nuclease genome editing system, either by transformation of the host cell with one or more expression vectors comprising all of the components, by integration of all of the components into the host cell genome, or by a mixture of transformation and integration of the components. In some aspects, the host cell encodes a Cas or Cas-like endonuclease and a gRNA.
Expression of the recombinase or endonuclease, including an endonuclease of a nuclease genome editing system, is under the control of an inducible promoter, i.e., a promoter which is activated under a particular physical or chemical condition or stimulus. In some aspects, the inducible promoter is thermally-regulated, chemically-regulated, IPTG regulated, glucose-regulated, arabinose inducible, T7 polymerase regulated, cold-shock inducible, pH inducible, or combinations thereof.
Provided herein is a recombinant cell comprising an expression vector as described herein that contains first and second target sequences for a first recombinase and one or more additional target sequences for one or more additional recombinases. In some aspects, the recombinant cell encodes the first recombinase and/or one or more of the one or more recombinases as described herein. In some aspects, the recombinant cell encodes one or more endonucleases as described herein. In some aspects, the recombinant cell encodes a nuclease genome editing system as described herein.
Provided herein is a method of producing a bacterial sequence-free vector comprising incubating a vector production system as described herein under suitable conditions for expression of the recombinase. In some aspects, the method further comprises incubating the vector production system under suitable conditions for expression of an endonuclease encoded by the recombinant cells. In some aspects, the method further comprises incubating the vector production system under suitable conditions for expression of a nuclease genome editing system encoded by the recombinant cells. In some aspects, the method further comprises harvesting the bacterial sequence-free vector.
Provided herein is a bacterial sequence-free vector produced by a method of producing a bacterial sequence-free vector as described herein.
Provided herein is a bacterial sequence-free vector comprising: (a) an expression cassette comprising a nucleic acid sequence of interest, and (b) one or more of: (i) a synthetic enhancer comprising a nucleic acid sequence at least about 90% identical to SEQ ID NO:12 located 5′ to another enhancer or a promoter in the expression cassette, (ii) a CMV enhancer located 5′ to a promoter in the expression cassette, (iii) a 5′UTR comprising an intron, wherein the 5′UTR is integrated in the expression cassette between a promoter and the nucleic acid sequence of interest, (iv) a vertebrate chromatin insulator integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal, (v) a WPRE integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal, (vi) a S/MAR integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal, or (vii) a DTS located 5′ to the expression cassette.
In some aspects, the bacterial sequence-free vector comprises a synthetic enhancer comprising a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:12 located 5′ to another enhancer or a promoter in the expression cassette. In some aspects, the bacterial sequence-free vector comprises a synthetic enhancer comprising the nucleic acid sequence of SEQ ID NO: 12 located 5′ to another enhancer or a promoter in the expression cassette. In some aspects, the synthetic enhancer comprises multiple contiguous copies of the nucleic acid sequence, such as, for example, 1, 2, 3, 4, 5, or more contiguous copies. In some aspects, the synthetic enhancer comprises 3 contiguous copies of the nucleic acid sequence. In some aspects, the synthetic enhancer comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:46. In some aspects, the synthetic enhancer comprises the nucleic acid sequence of SEQ ID NO:46. In some aspects, the synthetic enhancer is integrated at the 5′ end of a chicken β-actin promoter. In some aspects, a chimeric intron comprising a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:47 is integrated at the 3′ end of the chicken β-actin promoter and 5′ to the nucleic acid sequence of interest. In some aspects, a chimeric intron comprising the nucleic acid sequence of SEQ ID NO:47 is integrated at the 3′ end of the chicken β-actin promoter and 5′ to the nucleic acid sequence of interest.
In some aspects, the bacterial sequence-free vector comprises a CMV enhancer located 5′ to a promoter in the expression cassette. In some aspects, the CMV enhancer is integrated at the 3′ end of a synthetic enhancer comprising a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:12. In some aspects, the CMV enhancer is integrated at the 3′ end of a synthetic enhancer comprising the nucleic acid sequence of SEQ ID NO:12. In some aspects, the CMV enhancer is integrated at the 3′ end of multiple contiguous copies of the synthetic enhancer, such as, for example, at the 3′ end of 1, 2, 3, 4, 5, or more contiguous copies of the synthetic enhancer. In some aspects, the CMV enhancer is integrated at the 3′ end of 3 contiguous copies of the synthetic enhancer. In some aspects, the CMV enhancer is integrated at the 3′ end of a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:46. In some aspects, the CMV enhancer is integrated at the 3′ end of the nucleic acid sequence of SEQ ID NO:46. In some aspects, a CMV promoter is integrated at the 3′ end of the CMV enhancer and 5′ to the nucleic acid sequence of interest.
In some aspects, the bacterial sequence-free vector comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39 located 5′ to the nucleic acid sequence of interest. In some aspects, the bacterial sequence-free vector comprises the nucleic acid sequence of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39 located 5′ to the nucleic acid sequence of interest. In some aspects, a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39, or the nucleic acid sequence of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39, comprises all regulatory elements in the expression cassette located 5′ to the nucleic acid sequence of interest.
In some aspects, the bacterial sequence-free vector comprises a 5′UTR comprising an intron, wherein the 5′UTR (i.e., the 5′UTR comprising the intron) is integrated in the expression cassette between a promoter and the nucleic acid sequence of interest.
In some aspects, the 5′UTR is for improving transgene transcript splicing and translation from the bacterial sequence-free vector as compared to the same bacterial sequence-free vector lacking the 5′UTR.
In some aspects, the intron comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:1. In some aspects, the intron comprises the nucleic acid sequence of SEQ ID NO:1.
In some aspects, the 5′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:2. In some aspects, the 5′UTR comprises the nucleic acid sequence of SEQ ID NO:2.
In some aspects, the 5′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:4. In some aspects, the 5′UTR comprises the nucleic acid sequence of SEQ ID NO:4.
In some aspects, the 5′UTR further comprises a non-coding sequence integrated within the intron.
In some aspects, the intron is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:1, or comprises SEQ ID NO: 1, and the non-coding sequence is integrated between two of the nucleotides in the intron corresponding to any two nucleotides from positions 25 to 55 of SEQ ID NO:1.
In some aspects, the non-coding sequence is non-prokaryotic and non-viral. In some aspects, the non-coding sequence is eukaryotic. In some aspects, the non-coding sequence comprises an intron, a UCOE, a S/MAR, a SV40 enhancer sequence (e.g., one or more than one SV40 enhancer sequences, such as two, three, four, five or more SV40 enhancer sequences), a vertebrate chromatin insulator (e.g., cHS4), a WPRE, or any combination thereof.
In some aspects, the non-coding sequence comprises an S/MAR. In some aspects, the S/MAR is MAR-5, provided herein as SEQ ID NO:9.
In some aspects, the 5′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:3. In some aspects, the 5′UTR comprises SEQ ID NO:3.
In some aspects, the 5′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:5. In some aspects, the 5′UTR comprises SEQ ID NO:5.
In some aspects, the 5′UTR is integrated in the expression cassette between a chicken β-actin promoter and the nucleic acid sequence of interest.
In some aspects, the 5′UTR is integrated in the expression cassette between a CMV promoter and the nucleic acid sequence of interest.
In some aspects, the 5′UTR is integrated in the expression cassette between a promoter and the nucleic acid sequence of interest, wherein the promoter is integrated at the 3′ end of a CMV enhancer. In some aspects, the CMV enhancer is integrated at the 3′ end of a synthetic enhancer comprising a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:12. In some aspects, the CMV enhancer is integrated at the 3′ end of a synthetic enhancer comprising the nucleic acid sequence of SEQ ID NO: 12. In some aspects, the CMV enhancer is integrated at the 3′ end of multiple contiguous copies of the synthetic enhancer, such as, for example, at the 3′ end of 1, 2, 3, 4, 5, or more contiguous copies of the synthetic enhancer. In some aspects, the CMV enhancer is integrated at the 3′ end of 3 contiguous copies of the synthetic enhancer. In some aspects, the CMV enhancer is integrated at the 3′ end of a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:46. In some aspects, the CMV enhancer is integrated at the 3′ end of a nucleic acid sequence of SEQ ID NO:46.
In some aspects, the bacterial sequence-free vector comprises a polyadenylation signal integrated at the 3′ end of the nucleic acid sequence of interest. In some aspects, the polyadenylation signal comprises a Xenopus laevis beta-globin polyadenylation signal, a human beta-globin polyadenylation signal, or a hybrid Xenopus laevis and human beta-globin polyadenylation signal. In some aspects, the polyadenylation signal comprises multiple copies of a Xenopus laevis beta-globin polyadenylation signal, a human beta-globin polyadenylation signal, or a hybrid Xenopus laevis and human beta-globin polyadenylation signal, such as, for example, 1, 2, 3, 4, or 5 copies. In some aspects, the polyadenylation signal comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:13, SEQ ID NO: 14, or SEQ ID NO:15. In some aspects, the polyadenylation signal comprises the nucleic acid sequence of SEQ ID NO: 13, SEQ ID NO:14, or SEQ ID NO:15. In some aspects, a polyadenylic acid tail (i.e., poly(A) tail is located at the 3′ end of the polyadenylation signal. In some aspects, the poly(A) tail is 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, or more residues in length. In some aspects, the sequence comprising the polyadenylation signal and the poly(A) tail is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. In some aspects, the sequence comprising the polyadenylation signal and the poly(A) tail comprises SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.
In some aspects, the bacterial sequence-free vector comprises a vertebrate chromatin insulator in the expression cassette. In some aspects, the vertebrate chromatin insulator is cHS4. In some aspects, the vertebrate chromatin insulator is integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal as described herein. In some aspects, the vertebrate chromatin insulator is integrated within the intron of a 5′UTR as described herein.
In some aspects, the vertebrate chromatin insulator comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:8. In some aspects, the vertebrate chromatin insulator comprises SEQ ID NO:8.
In some aspects, the vertebrate chromatin insulator is for improving establishment (i.e., transfection efficiency) of a bacterial sequence-free vector as compared to the same bacterial sequence-free vector without the vertebrate chromatin insulator.
In some aspects, the bacterial sequence-free vector comprises a WPRE in the expression cassette. In some aspects, the WPRE is integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal as described herein. In some aspects, the WPRE is integrated in the expression cassette at the 3′ end of a S/MAR as described herein and the 5′ end of a polyadenylation signal as described herein. In some aspects, the WPRE is integrated within the intron of a 5′UTR as described herein.
In some aspects, the WPRE comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:11. In some aspects, the WPRE comprises SEQ ID NO:11.
In some aspects, the WPRE improves expression of the transgene from the bacterial sequence-free vector as compared to the same bacterial sequence-free vector lacking the WPRE.
In some aspects, the bacterial sequence-free vector comprises an S/MAR in the expression cassette. In some aspects, the S/MAR is integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal. In some aspects, the S/MAR is integrated in the expression cassette at the 3′ end of a nucleic acid sequence of interest and the 5′ end of a WPRE as described herein. In some aspects, the S/MAR is integrated within the intron of a 5′UTR as described herein.
In some aspects, the S/MAR is MAR-3, MAR-4, or MAR-5. In some aspects, the S/MAR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:9. In some aspects, the S/MAR comprises SEQ ID NO:9.
In some aspects, the S/MAR is human CSP-B MAR or CSP-C MAR. In some aspects, the S/MAR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:10. In some aspects, the S/MAR comprises SEQ ID NO:10.
In some aspects, the S/MAR is for improving expression levels, stability, and/or durability of the bacterial sequence-free vector (e.g., by episomal maintenance and replication, such as expansion and partition of the vector to daughter cells, and/or by preventing epigenetic silencing) as compared to the same bacterial sequence-free vector lacking the S/MAR.
In some aspects, the bacterial sequence-free vector comprising any one of more of (b)(i)-(b)(v) as described above (i.e., without a DTS) further comprises an enhancer sequence flanking each side of the expression cassette. In some aspects, the enhancer sequence flanking each side of the expression cassette is at least two enhancer sequences flanking each side of the expression cassette. In some aspects, the enhancer sequence is a SV40 enhancer sequence.
In some aspects, the bacterial sequence-free vector comprises a DTS. In some aspects, the DTS is located 5′ to the expression cassette. In some aspects, the DTS is a SV40 enhancer sequence. In some aspects, the DTS is cell-specific. In some aspects, the DTS is specific for smooth muscle cells, embryonic stem cells, type II pneumonocytes, endothelial cells, or osteoblasts.
In some aspects, a bacterial sequence-free vector as described herein further comprises a UCOE in the expression cassette. In some aspects, the UCOE is located 5′ to the promoter or any enhancer in the expression cassette. In some aspects, the UCOE is integrated within the intron of a 5′UTR as described herein.
In some aspects, the UCOE is A2UCOE. In some aspects, the UCOE comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:6. In some aspects, the UCOE is SEQ ID NO:6.
In some aspects, the UCOE is SRF-UCOE. In some aspects, the UCOE comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:7. In some aspects, the UCOE is SEQ ID NO:7.
In some aspects, the UCOE improves expression of the transgene from the bacterial sequence-free vector as compared to the same bacterial sequence-free vector lacking the UCOE.
In some aspects, the bacterial sequence-free vector comprises Enhancer-1 in the expression cassette. In some aspects, Enhancer-1 is integrated 5′ to the promoter or any other enhancer in the expression cassette. In some aspects, Enhancer-1 is integrated between the 3′ end of a UCOE and the 5′ end of a CMV enhancer. In some aspects, Enhancer-1 comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:12. In some aspects, Enhancer-1 is SEQ ID NO: 12.
In some aspects, the bacterial sequence-free vector comprises a CMV, EF1, SV40, CAG, Rho, VDM2, HCR, or HLP promoter, or variant thereof, in the expression cassette. In some aspects, the bacterial sequence-free vector comprises a CMV promoter variant in the expression cassette.
In some aspects, the bacterial sequence-free vector comprises an EF1-alpha promoter in the expression cassette. In some aspects, the bacterial sequence-free vector comprises a CMV enhancer and an EF1-alpha promoter in the expression cassette.
In some aspects, the bacterial sequence-free vector comprises a 3′UTR in the expression cassette comprising two copies of a beta-globin polyadenylation signal. In some aspects, the 3′UTR is integrated 3′ to the nucleic acid sequence of interest.
In some aspects, the 3′UTR comprises two copies of a Xenopus laevis beta-globin polyadenylation signal. In some aspects, the 3′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 13. In some aspects, the 3′UTR is SEQ ID NO:13.
In some aspects, the 3′UTR comprises two copies of a human beta-globin polyadenylation signal. In some aspects, the 3′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 14. In some aspects, the 3′UTR is SEQ ID NO:14.
In some aspects, the 3′UTR comprises one copy of a Xenopus laevis beta-globin polyadenylation signal and one copy of a human beta-globin polyadenylation signal. In some aspects, the 3′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:15. In some aspects, the 3′UTR is SEQ ID NO:15.
In some aspects, the 3′UTR further comprises a poly(A) tail comprising 100 to 120 adenine nucleotides, i.e., 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 adenine nucleotides.
In some aspects, the 3′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:16. In some aspects, the 3′UTR is SEQ ID NO:16.
In some aspects, the 3′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:17. In some aspects, the 3′UTR is SEQ ID NO:17.
In some aspects, the 3′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:18. In some aspects, the 3′UTR is SEQ ID NO:18.
The nucleic acid sequence of interest of a bacterial sequence-free vector as described herein includes any of the nucleic acid sequences described herein with respect to the expression vectors for producing the bacterial sequence-free vectors.
In some aspects, a bacterial sequence-free vector as described herein comprises a Cas endonuclease target sequence (i.e., a sequence homologous to a gRNA targeting sequence) located 5′ and 3′ to the nucleic acid sequence of interest, wherein a target site for gene editing (e.g., a target site in a chromosome) comprises the same Cas endonuclease target sequence.
In some aspects, a bacterial sequence-free vector as described herein comprises a CRISPR-Cas system. In some aspects, the bacterial sequence-free vector comprises a tRNA-gRNA polycistron flanking each side of a sequence encoding a Cas endonuclease (e.g., an immunosilenced Cas9-B2). In some aspects, the bacterial sequence-free vector comprises a 5′UTR (e.g., 5′UTR1) as described herein comprising the tRNA-gRNA polycistron in an intron. In some aspects, the bacterial sequence-free vector comprises a chimeric intron as described herein comprising the tRNA-gRNA polycistron. In some aspects, an EF1-alpha promoter as described herein comprises the tRNA-gRNA polycistron in an inherent intron. In some aspects, a polyadenylation signal or 3′UTR as described herein comprises a tRNA-gRNA polycistron. In some aspects, a nucleic acid sequence of interest and a self-restricting CRISPR-Cas system as described herein are located on a single bacterial sequence-free vector as described herein. In the latter aspects, the sequences comprising the self-restricting CRISPR-Cas system are located 5′ to the sequence comprising the nucleic acid sequence of interest flanked by homology arms.
A bacterial sequence-free vector as described herein can contain any combination of the above modifications. In some aspects, the combination provides a synergistic effect.
In some aspects, the bacterial sequence-free vector is a circular covalently closed vector.
In some aspects, the bacterial sequence-free vector is a linear covalently closed vector.
Provided herein is a recombinant cell comprising a bacterial sequence-free vector as disclosed herein.
Improvements and modifications described above can also be applied to other expression vectors such as, but not limited to, expression vectors that are utilized for direct gene expression rather than production of bacterial sequence-free vectors. In some aspects, the nucleic acid sequences described herein are provided as DNA sequences, and the expression vectors are DNA expression vectors. In some aspects, the nucleic acid sequences described herein are provided as RNA sequences, and the expression vectors are RNA expression vectors. RNA sequences can correspond to the DNA sequence provided as any SEQ ID NO herein or can correspond to the DNA sequence that is complementary to the DNA sequence provided as any SEQ ID NO herein.
Provided herein is a polynucleotide comprising any combination of nucleic acid sequences as described herein.
Provided herein is a polynucleotide comprising a nucleic acid sequence of: an intron, a 5′UTR comprising an intron, and/or a 3′UTR as described herein.
Provided herein is a polynucleotide comprising a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 1, 2, 3, 5, 13, 14, 15, 16, 17, or 18. In some aspects, the polynucleotide comprises 100 to 120 adenine nucleotides at the 3′ end of the nucleic acid sequence. In some aspects, the polynucleotide comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs:13, 14, or 15, and 100 to 120 adenine nucleotides at the 3′ end of the nucleic acid sequence. In some aspects, the polynucleotide comprises the nucleic acid sequence of any one of SEQ ID NOs: 1, 2, 3, 5, 13, 14, 15, 16, 17, or 18.
Provided herein is an expression vector comprising one or more of the polynucleotides described herein. In some aspects, the expression vector comprises a polynucleotide comprising a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 1, 2, 3, 5, 13, 14, 15, 16, 17, or 18. In some aspects, the polynucleotide comprises 100 to 120 adenine nucleotides at the 3′ end of the nucleic acid sequence. In some aspects, the polynucleotide comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 13, 14, or 15, and 100 to 120 adenine nucleotides at the 3′ end of the nucleic acid sequence. In some aspects, the expression vector comprises a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 1, 2, 3, 5, 13, 14, 15, 16, 17, or 18. In some aspects, the expression vector comprises a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 2, 3, or 5, and (a) a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 13, 14, 15, 16, 17, or 18, or (b) a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 13, 14, or 15 and 100 to 120 adenine nucleotides at the 3′ end of the nucleic acid sequence.
Provided herein is an expression vector comprising: a 5′UTR comprising an intron, wherein the 5′UTR is integrated in the expression cassette between a promoter and a nucleic acid sequence of interest, and/or a 3′UTR comprising two copies of a beta-globin polyadenylation signal integrated in the expression cassette 3′ to the nucleic acid sequence of interest.
In some aspects, the 5′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:2. In some aspects, the 5′UTR comprises the nucleic acid sequence of SEQ ID NO:2.
In some aspects, the 5′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:4. In some aspects, the 5′UTR comprises the nucleic acid sequence of SEQ ID NO:4.
In some aspects, the 5′UTR further comprises a non-coding sequence integrated within the intron.
In some aspects, the intron is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:1, or comprises SEQ ID NO:1, and the non-coding sequence is integrated between two of the nucleotides in the intron corresponding to any two nucleotides from positions 25 to 55 of SEQ ID NO:1.
In some aspects, the non-coding sequence is non-prokaryotic and non-viral. In some aspects, the non-coding sequence is a eukaryotic sequence. In some aspects, the non-coding sequence comprises an intron, a UCOE, an S/MAR, an SV40 enhancer sequence (e.g., one or more than one SV40 enhancer sequences, such as two, three, four, five or more SV40 enhancer sequences), a vertebrate chromatin insulator (e.g., cHS4), a WPRE, or any combination thereof.
In some aspects, the non-coding sequence is an S/MAR. In some aspects, the S/MAR is MAR-5, provided herein as SEQ ID NO:9.
In some aspects, the 5′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:3. In some aspects, the 5′UTR comprises SEQ ID NO:3.
In some aspects, the 5′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:5. In some aspects, the 5′UTR comprises SEQ ID NO:5.
In some aspects, the 3′UTR comprises two copies of a Xenopus laevis beta-globin polyadenylation signal. In some aspects, the 3′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:13. In some aspects, the 3′UTR is SEQ ID NO:13.
In some aspects, the 3′UTR comprises two copies of a human beta-globin polyadenylation signal. In some aspects, the 3′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:14. In some aspects, the 3′UTR is SEQ ID NO:14.
In some aspects, the 3′UTR comprises one copy of a Xenopus laevis beta-globin polyadenylation signal and one copy of a human beta-globin polyadenylation signal. In some aspects, the 3′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 15. In some aspects, the 3′UTR is SEQ ID NO:15.
In some aspects, the 3′UTR further comprises a poly(A) tail comprising 100 to 120 adenine nucleotides, i.e., 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 adenine nucleotides.
In some aspects, the 3′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:16. In some aspects, the 3′UTR is SEQ ID NO:16.
In some aspects, the 3′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:17. In some aspects, the 3′UTR is SEQ ID NO:17.
In some aspects, the 3′UTR comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:18. In some aspects, the 3′UTR is SEQ ID NO:18.
Provided herein is an expression vector comprising a synthetic enhancer comprising a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:12. In some aspects, the expression vector comprises a synthetic enhancer comprising the nucleic acid sequence of SEQ ID NO: 12. In some aspects, the synthetic enhancer comprises multiple contiguous copies of the nucleic acid sequence, such as, for example, 1, 2, 3, 4, 5, or more contiguous copies. In some aspects, the synthetic enhancer comprises 3 contiguous copies of the nucleic acid sequence. In some aspects, the synthetic enhancer comprises a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:46. In some aspects, the synthetic enhancer comprises the nucleic acid sequence of SEQ ID NO:46.
Provided herein is an expression vector comprising a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39. In some aspects, the expression vector comprises the nucleic acid sequence of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39. In some aspects, a nucleic acid sequence at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39, or the nucleic acid sequence of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39, comprises all regulatory elements in an expression cassette located 5′ to a nucleic acid sequence of interest in the expression vector.
Provided herein is a composition comprising an expression vector or bacterial sequence-free vector as described herein.
A variety of methods are known in the art and are suitable for introduction of nucleic acids into a cell. Examples include, but are not limited to, electroporation, calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, liposome mediated transfer, microinjection, microprojectile mediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG), and the like), or cell fusion.
Nanoparticle carriers such as liposomes, micelles, and polymeric nanoparticles have been investigated for improving bioavailability and pharmacokinetic properties of therapeutics via various mechanisms, for example, the enhanced permeability and retention (EPR) effect.
Further improvement can be achieved by conjugation of targeting ligands onto nanoparticles to achieve selective delivery to a target cell. For example, receptor-targeted nanoparticle delivery has been shown to improve therapeutic responses both in vitro and in vivo. Targeting ligands that have been investigated include folate, transferrin, antibodies, peptides, and aptamers. Additionally, multiple functionalities can be incorporated into the design of nanoparticles, e.g., to enable imaging and to trigger intracellular drug release.
In some aspects, the composition further comprises a delivery agent. In some aspects, the delivery agent is a nanoparticle. In some aspects, the delivery agent is selected from the group consisting of liposomes, non-lipid polymeric molecules, endosomes, and any combination thereof.
In some aspects, the delivery agent (e.g., a nanoparticle) comprises a targeting ligand.
In some aspects, the composition further comprises a physiologically acceptable carrier, excipient, or stabilizer. See, e.g., Remington: The Science and Practice of Pharmacy, 22nd ed. (2013). Acceptable carriers, excipients, or stabilizers can include those that are nontoxic to a subject. In some aspects, the composition or one or more components of the composition are sterile. A sterile component can be prepared, for example, by filtration (e.g., by a sterile filtration membrane) or by irradiation (e.g., by gamma irradiation).
In some aspects, the composition comprising an expression vector or bacterial sequence-free vector as described herein is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.
An excipient of the present invention can be described as a “pharmaceutically acceptable” excipient when added to a pharmaceutical composition, meaning that the excipient is a compound, material, composition, salt, and/or dosage form which is, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problematic complications over the desired duration of contact commensurate with a reasonable benefit/risk ratio. In some aspects, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized international pharmacopeia for use in animals, and more particularly in humans. Various excipients can be used. In some aspects, the excipient can be, but is not limited to, an alkaline agent, a stabilizer, an antioxidant, an adhesion agent, a separating agent, a coating agent, an exterior phase component, a controlled-release component, a solvent, a surfactant, a humectant, a buffering agent, a filler, an emollient, or combinations thereof. Excipients in addition to those discussed herein can include excipients listed in, though not limited to, Remington: The Science and Practice of Pharmacy, 22nd ed. (2013). Inclusion of an excipient in a particular classification herein (e.g., “solvent”) is intended to illustrate rather than limit the role of the excipient. A particular excipient can fall within multiple classifications.
A pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration. Exemplary routes of administration include enteral, topical, parenteral, oral, pulmonary, intranasal, intravenous, epidermal, transdermal, subcutaneous, intramuscular, or intraperitoneal administration, or inhalation. “Parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection or infusion, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrapleural, and intrasternal injection and infusion, as well as in vivo electroporation. In some aspects, the formulation is administered via a non-parenteral route, in some aspects, orally. Other non-parenteral routes include a topical, epidermal, or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically.
In some aspects, the pharmaceutical composition is lyophilized.
Provided herein is a method of treating a disease or disorder in a subject in need thereof, comprising administering an expression vector, bacterial sequence-free vector, or pharmaceutical composition as described herein to the subject.
The expression vector, bacterial sequence-free vector, or composition can be administered to a subject by any route of administration that is effective for treating the disease or disorder.
In some aspects, the administering is by enteral, topical, parenteral, oral, pulmonary, intranasal, intravenous, epidermal, transdermal, subcutaneous, intramuscular, intrathecal, or intraperitoneal administration, inhalation, or cerebrospinal fluid (CSF)-based delivery via intracerebroventricular (ICV) injection, cisterna magna administration (ICM), or lumbar intrathecal puncture (LIT).
In some aspects, the administering is by parenteral or non-parenteral administration.
In some aspects, the parenteral administration is by injection or infusion.
In some aspects, the parenteral administration is by intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, retroorbital, intracerebroventricular, subarachnoid, intraspinal, epidural, intrapleural, or intrasternal injection or infusion, or by in vivo electroporation, nucleofection, microbubble, or ultrasound.
In some aspects, the non-parenteral administration is oral, topical, epidermal, mucosal, intranasal, vaginal, rectal, or sublingual.
In some aspects, the administering is by oral, pulmonary, intranasal, intravenous, epidermal, transdermal, subcutaneous, intramuscular, or intraperitoneal administration, or by inhalation.
In some aspects, the administering is by oral, nasal, or pulmonary administration. In some aspects, the administering is by nasal administration.
Administering can be performed, for example, once, a plurality of times, and/or over one or more extended periods. In some aspects, the administering is one time, two times (e.g., a first administration followed by a second administration about 1, about 2, about 3, about 4 or more weeks later), once about every week, once about every month, once about every 2 months, once about every 3 months, once about every 4 months, once about every 6 months, once about every year, or once about every decade.
Provided herein is a method of gene editing comprising inserting a nucleic acid sequence of interest from an expression vector, bacterial sequence-free vector, or pharmaceutical composition as described herein into a target site for gene editing.
In some aspects, the inserting is by non-homologous end joining.
In some aspects, the inserting is by homology directed repair. In some aspects, the nucleic acid sequence of interest is flanked by 5′ and 3′ homology arms as described herein.
In some aspects, the nucleic acid sequence of interest is homologous to the target site for gene editing and comprises one or more nucleotide insertions, deletions, inversions, or rearrangements as compared to the target site.
In some aspects, the nucleic acid sequence of interest is non-homologous to the target site for gene editing.
In some aspects, the nucleic acid sequence of interest restores a missing function, corrects an abnormal function, or provides an additional function associated with the target site for gene editing.
In some aspects, the nucleic acid sequence of interest is for knockout of gene expression associated with the target site for gene editing.
In some aspects, the method of gene editing is a method of treating a disease or disorder in a subject in need thereof.
In some aspects, the nucleic acid sequence of interest is for in vivo gene editing.
In some aspects, the nucleic acid sequence of interest is for in vitro gene editing.
In some aspects, the nucleic acid sequence of interest is for ex vivo gene editing (e.g., cell therapy, such as CAR T cell therapy).
In some aspects, the method is an in vitro method. In some aspects, the in vitro method further comprises administering the expression vector, bacterial sequence-free vector, or pharmaceutical composition to cells (e.g., for in vitro or ex vivo gene editing). In some aspects, the in vitro method further comprises administering an endonuclease for gene editing, or a genome editing system or components thereof (e.g., Cas endonuclease and gRNA for a CRISPR-Cas system) to the cells. In some aspects, the genome editing system is a CRISPR-Cas, TALEN, ZFN, or meganuclease gene editing system.
In some aspects, the method is an in vivo method. In some aspects, the in vivo method further comprises administering the expression vector, bacterial sequence-free vector, or pharmaceutical composition to a subject. In some aspects, the in vivo method further comprises administering an endonuclease for gene editing, or a genome editing system or components thereof (e.g., Cas endonuclease and gRNA for a CRISPR-Cas system) to the subject. In some aspects, the genome editing system is a CRISPR-Cas, TALEN, ZFN, or meganuclease gene editing system.
The endonuclease for gene editing, or the genome editing system or components thereof, can be administered by any methods described herein or as known in the art for administering nucleic acid sequences and/or polypeptides to cells or subjects, including through electroporation or vectors as applicable to the administration. For example, in aspects comprising a CRISPR-Cas system, RNA encoding Cas and/or gRNA can be administered, Cas and/or gRNA can be directly administered, bacterial sequence-free vectors or expression vectors as described herein can be administered that encode Cas and/or gRNA, or any other suitable vector known in the art can be administered that encode Cas and/or gRNA.
In some aspects, the nucleic acid of interest is provided in a linear covalently closed bacterial sequence-free vector (i.e., msDNA) as described herein. In some aspects, use of the linear covalently closed bacterial sequence-free vector in gene editing avoids any undesired non-homologous end joining because the ends of the bacterial sequence-free vector are closed and non-reactive with double strand breaks. In some aspects, use of the linear covalently closed bacterial sequence-free vector in gene editing enhances homology-directed repair. In some aspects, the recombination rate for homology-directed repair is higher when the nucleic acid sequence of interest is provided by a linear covalently closed bacterial sequence-free vector as described herein than when the nucleic acid sequence of interest is provided by a circular supercoiled vector.
The following examples are offered by way of illustration and not by way of limitation.
A polygenic expression vector was prepared by replacing the eGFP coding sequence of a parent ministring expression vector (Mediphage Bioceuticals, Inc., Toronto, CA, U.S. Pat. Nos. 9,290,778 and 9,862,954), pGL2-SS*-CAG-eGFP-BGpA-SS*, with an expression cassette encoding enhanced green fluorescent protein (eGFP) and the NanoLuc® luciferase reporter modified with a secretion sequence for extracellular expression (NLuc, Promega Corporation) between the two specialized Super Sequence (SS*) sites of the parent vector.
The expression cassette of the parent vector and the polygenic vector contained a CAG promoter, which is a synthetic promoter that includes a cytomegalovirus (CMV) enhancer, a promoter from chicken β-actin, and a chimeric intron.
A map of the polygenic expression vector is shown in
A second polygenic expression vector was prepared by cloning the same eGFP and Nluc sequences along with a 5′UTR into the pcDNA3.1 vector (Thermo Fisher Scientific). A map of the expression vector is shown in
Adherent human embryonic kidney 293 (HEK293) cells were seeded in a 24-well plate at 1×105 cells/well.
A complex of expression vector (1 μg) and lipofectamine (3 μL) was prepared and incubated using standard operating procedures for each of (1) pGL2-SS*-CAG-SecNLuc-2A-eGFP-BGpA-SS*, (2) pcDNA-CMV-5′UTR-SecNLuc-P2A-eGFP-bGHpA, and (3) pGL2-SS*-CAG-eGFP-BGpA-SS*.
The three complexes were used to separately transfect HEK293 cells via electroporation in individual wells, which were then incubated for 48 hours. HEK293 cells in other wells were treated with 3 μL lipofectamine containing no plasmid as a negative control.
Cells were evaluated for cytoplasmic GFP expression and luciferase expression 48 hours after transfection.
Cytoplasmic GFP expression was used as a measure of transfection efficiency and gene expression levels by the polygenic expression vectors. Expression was evaluated by fluorescent microscopy, and mean GFP expression/intensity of the experimental expression vectors (pGL2-SS*-CAG-SecNLuc-2A-eGFP-BGpA-SS* and pcDNA-CMV-5′UTR-SecNLuc-P2A-eGFP-bGHpA) was measured relative to the negative control (cells treated with lipofectamine and no plasmid) and the positive control (pGL2-SS*-CAG-eGFP-BGpA-SS*), also referred to herein as parental plasmid CAG-GFP, i.e., PP-CAG-GFP).
Live imaging of fluorescent cells under auto exposure mode showed that the experimental expression vectors produced GFP, with the chimeric intron of pGL2-SS*-CAG-SecNLuc-2A-eGFP-BGpA-SS*and the 5′UTR of pcDNA-CMV-5′UTR-SecNLuc-P2A-eGFP-bGHpA having similar expression. See
Luciferase expression was evaluated by measuring the intensity of secreted luciferase in the media of transfected cells and negative control cells using the Nano-Glo® Luciferase Assay System (Promega) according to manufacturer protocols. Both experimental expression vectors expressed luciferase. See
A polygenic expression vector was prepared by cloning a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) between the sequence encoding eGFP and BGpA in the expression vector of
Another polygenic expression vector was prepared that contains a CMV enhancer/promoter and an engineered 5′UTR containing an internal minimal intron sequence (i.e., 5′UTR1, SEQ ID NO:2) in place of the CAG promoter in
A further polygenic expression vector was prepared that contains a CMV enhancer/promoter and an engineered 5′UTR containing an intron with an integrated MAR-5 (i.e., 5′UTR2, SEQ ID NO:5) in place of the CAG promoter in
Adherent HEK293 cells were detached, resolved in electroporation media, and counted at 1×106 cells/tube.
The expression vector (1 μg) was prepared and incubated with cells using standard operating procedures for each of (1) pGL2-SS*-CAG-SecNLuc-2A-eGFP-BGpA-SS* (see Example 1), (2) pGL2-SS*-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS*, (3) pGL2-SS*-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA-SS*, and (4) pGL2-SS*-CMV-UTR2-SecNLuc-2A-eGFP-WPRE-BGpA-SS*.
HEK293 cells electroporated with puc57 plasmid lacking a mammalian expression cassette served as a negative control.
After electroporation, HEK293 cells were seeded and adhered to wells at 3×105 cells/well.
On days 2, 6, 10, 14, 17, 20, 27, and 34 after electroporation, luciferase expression was evaluated by measuring the intensity of secreted luciferase in 20 μL of cell culture media in triplicate for each of the four transfections and the negative control using the Nano-Glo® Luciferase Assay System (Promega) according to manufacturer protocols. Luciferase activity was measured using a BioTek® plate reader and displayed in Relative Luminometer Units (RLU). Statistical analysis of luciferase activity was performed by Student's T-test. See
Luciferase expression was detected throughout the duration of the experiment from cells transfected with any of the four expression vectors. pGL2-SS*-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS*, pGL2-SS*-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA-SS*, and pGL2-SS*-CMV-UTR2-SecNLuc-2A-eGFP-WPRE-BGpA-SS* all showed significantly higher luciferase expression as compared to pGL2-SS*-CAG-SecNLuc-2A-eGFP-BGpA-SS*, with pGL2-SS*-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA-SS* showing the highest enhancement of expression.
HEK293 cells were transfected with the four expression vectors or the puc57 plasmid as a negative control, as described in part B of this example. Cells were passaged weekly for five passages. At the time of cell passaging, cells were re-seeded at ⅙ of the original cell density for passage numbers 1-3, and 1/10 of the original cell density for passage numbers 4-5. For each cell passage, secreted luciferase expression was measured 6-8 days after cell re-seeding as described in part B of this example. See
Luciferase expression was detected from cells transfected with any of the four expression vectors at each passage number, showing that the vectors were passed down to daughter cells with durable expression of luciferase. pGL2-SS*-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS*, pGL2-SS*-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA-SS*, and pGL2-SS*-CMV-UTR2-SecNLuc-2A-eGFP-WPRE-BGpA-SS* all showed significantly higher luciferase expression at each passage number as compared to pGL2-SS*-CAG-SecNLuc-2A-eGFP-BGpA-SS*, with pGL2-SS*-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA-SS* showing the highest enhancement of expression.
In a subsequent study, msDNA expansion to daughter cells with durable expression of luciferase was also observed.
Briefly, msDNA was produced from pGL2-SS*-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA-SS* in an inducible E. coli vector production system using methods described herein and in U.S. Pat. Nos. 9,290,778 and 9,862,954. Separate complexes with lipofectamine were prepared with (1) the msDNA (i.e., msDNA-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA) (2) the parental plasmid (i.e., pGL2-SS*-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA-SS*), and (3) a conventional plasmid with a luciferase expression cassette (i.e., pcDNA-CMV-5′UTR-SecNLuc-P2A-eGFP-bGHpA). HEK293 cells were separately transfected with the vectors via electroporation in individual wells for a total of 0.25 pmol vector/well. Cells were passaged 7 times, with a 10-fold cell dilution at each passage. Relative luciferase intensity was determined on days 8, 15, 24, 31, 38, 45, and 52 for passage numbers 1, 2, 3, 4, 5, 6, and 7, respectively.
As shown in
D. Vector Expansion to Daughter Cells and eGFP Expression
Cells transfected with pGL2-SS*-CAG-SecNLuc-2A-eGFP-BGpA-SS* (pGL2-SecNLuc-eGFP) or pGL2-SS*-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA-SS* (5′UTR1+WPRE) and passaged according to part C of this example were analyzed for eGFP expression.
Imaging was performed 6 to 8 days after cell passaging for each passage number. Live cell imaging was performed using a BioTek® Cytation™ 5 plate reader. See
One representative image was taken from each triplicate well for each expression vector, and eGFP expressing cells (GFP) were quantified by manual cell counting using ImageJ computer software. Statistical analysis of GFP′ cells was performed using a Student's t-test. See
ImageJ software was used to manually select each GFP′ cell and measure the Mean Fluorescence Intensity (MFI) for each cell based on pixel intensity. To calculate the final MFI value for each cell, the following formula was used (Final MFI=Cell MFI−Background MFI). MFI measurements were obtained for at least 50 cells from each of the 3 images taken for each treatment group. All MFI measurements were then pooled and used to generate a dot plot. Statistical analysis was performed using a Student's t-test. See
Studies were conducted to assess targeted delivery of msDNA to the liver, retina, and brain. For each target tissue, different routes of administration (ROAs), doses, dosing regimens, and delivery techniques were evaluated. Secreted luciferase expression kinetics, cytoplasmic eGFP expression levels, and transfection efficiency (TE) were evaluated. In addition, tolerability to the msDNA was evaluated after single or multiple injections by physiological assessment, tissue morphology analysis, plasma cytokine assay, and liver toxicity analysis.
Across all delivery techniques, msDNA showed strong efficacy and tolerability profiles in the brain and liver tissues via multiple intracerebroventricular (ICV) or hydrodynamic injections (HDI) and intravenous (IV) injections, respectively. Adult mice treated with msDNA showed sustained secreted luciferase levels (>108 RLU/mg protein) after a single IV injection. The msDNA showed durable (>100 days) expression in the liver tissue after a single IV injection. Significant biodistribution to deep tissue regions was also demonstrated, with 80% to 97% TE in brainstem, cerebellum, cortex, and thalamus. The triple ICV injections with the nanocarrier-msDNA complex did not show any side effects.
1. Expression of Luciferase from a Single High Dose, 2 mg/kg (50 μg), Hydrodynamic Injection of Carrier-Free Naked Plasmid
C57BL/6J male wild-type adult 8-12 weeks old mice were administered a single high dose of 2 mg/kg (50 μg) of carrier-free pcDNA-CMV-5′UTR-SecNLuc-P2A-eGFP-bGHpA (positive control with no supersequence), pGL2-SS*-CAG-SecNLuc-2A-eGFP-BGpA-SS*, pGL2-SS*-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA-SS*, or pGL2-SS*-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS* by hydrodynamic injection (HDI) via the tail vein. The plasma of the treated mice was collected on days 1, 3, 7, 10, 15, 22, 28, 42, and 56 after HDI to examine luciferase gene expression. On day 1 post-vector administration, all mice exhibited high levels of luciferase expression (108-109 RLU per mg of plasma protein). On day-7, the pCAGLuc and the pCAGLuc-WPRE treated mice produced 107-108 RLU/mg of plasma protein, but the pGSNLuc-WPRE treated mice yielded lower luciferase levels (˜106 RLU/mg protein). After 8-weeks post-vector administration, all mice exhibited low levels of luciferase expression (around 105 RLU/mg protein). The rapid drop of luciferase levels may have resulted from humoral or cell-mediated immune responses induced in the plasmid treated mice. See
2. Expression of Luciferase from a Single Low Dose, 0.2 mg/kg (5 μg), Hydrodynamic Injection of Carrier-Free Naked Plasmid
To test dose response of plasmid DNA following nonviral gene delivery in animal models, C57BL/6J male wild-type adult 8-12 weeks old mice were administered a single low dose of 0.2 mg/kg (5 μg) of carrier-free pcDNA-CMV-5′UTR-SecNLuc-P2A-eGFP-bGHpA (positive control with no supersequence, 2 mice), pGL2-SS*-CAG-SecNLuc-2A-eGFP-BGpA-SS* (2 mice), or pGL2-SS*-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS* (2 mice) by HDI via the tail vein. An additional 2 mice were not injected and served as a negative control. The plasma of the mice was collected on days 1, 3, 7, 10, 15, 22, 28, 42, and 56 after HDI to examine luciferase gene expression. The mice treated with pGL2-SS*-CAG-SecNLuc-2A-eGFP-BGpA-SS* and pGL2-SS*-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS* showed sustained high levels of luciferase expression (107-108 RLU/mg protein) more than 8 weeks post-vector administration and more than 100-fold higher expression than the conventional control plasmid having an isogenic expression cassette but with no supersequence (SS). See
In vivo whole body bioluminescence imaging (BLI) with IVIS was conducted by injecting a 1:5 dilution of fluorofurimazine (FFz) intraperitoneally 24 hours after HDI of the vectors. The BLI was shown to correlate with the level of luciferase in the plasma samples (data not shown).
3. Expression of Luciferase from a Single Low Dose, 0.2 mg/kg (5 μg), Hydrodynamic Injection of Carrier-Free Naked msDNA
msDNAs were produced from pGL2-SS*-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS* and pGL2-SS*-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA-SS* in an inducible E. coli vector production system using methods described herein and in U.S. Pat. Nos. 9,290,778 and 9,862,954.
C57BL/6J male wild-type adult 8-12 weeks old mice were administered a single low dose of 0.2 mg/kg (5 μg) of carrier-free pcDNA-CMV-5′UTR-SecNLuc-P2A-eGFP-bGHpA (positive control, 5 mice), msDNA-CAG-SecNLuc-2A-eGFP-WPRE-BGpA (5 mice), or msDNA-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA (5 mice) by hydrodynamic injection (HDI) via the tail vein. An additional 2 mice were not injected and served as a negative control. The plasma of the treated mice was collected on days 1, 3, 7, 10, 15, 22, 28, 42, and 56 after HDI to examine luciferase gene expression.
Similarly to plasmid treated mice, the msDNA-CAG-SecNLuc-2A-eGFP-WPRE-BGpA treated mice produced sustained high levels of luciferase expression (107-108 RLU/mg protein) more than 8 weeks post-vector administration, whereas the luciferase expression in msDNA-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA treated mice dropped to low levels (˜106 RLU/mg protein) in less than one month. The rapid drop of luciferase expression in msDNA-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA treated mice was likely due to silencing of the CMV promoter in hepatocytes.
Luciferase gene expression was confirmed via whole body live imaging with IVIS.
Table 1, below, provides data from individual mice on days 1, 7, and 28 for luciferase expression in plasma samples (RLU/mg protein) and as detected by BLI (photons/see).
As shown in
The data show that nonviral delivery with msDNA in mice was highly efficient and the resulting gene expression was stable for more than two months.
4. Expression of eGFP from a Single Low Dose, 0.2 mg/kg (5 μg), Hydrodynamic Injection of Carrier-Free Naked msDNA
Intracellular cytoplasmic eGFP expression levels were evaluated by ELISA. Briefly, liver samples were collected from mice at 56 days after HDI with the single low dose of 0.2 mg/kg (5 μg) of the vectors as described in part 3 and homogenized for protein extraction. Total protein concentrations were determined from the liver lysates. GFP protein levels were then analyzed by ELISA.
As evident by comparing the data in
As shown in
5. Expression of msDNA in Liver after Low Dose Single Intravenous Injection and Tolerability Profile
C57BL/6J male wild-type adult 8-12 weeks old mice were administered 0.3 mg/kg pcDNA-CMV-5′UTR-SecNLuc-P2A-eGFP-bGHpA (positive control with no supersequence), msDNA-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA, pGL2-SS*-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA-SS*, msDNA-CAG-SecNLuc-2A-eGFP-WPRE-BGpA, or pGL2-SS*-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS* lipoplexed with a lipid nanoparticle carrier through a single intravenous tail vein injection. The carrier also served as a negative vehicle control.
In vivo whole body bioluminescence imaging (BLI) with IVIS was conducted as described above on days 1, 3, 10, 30, 58, 92, 119, and 174 after the single IV injection of the vectors. As shown in
Serum alanine aminotransferase (ALT) level, liver cytotoxicity, and cytokine responses also were evaluated following injection of the vectors. Precursor plasmid and msDNA containing the CAG promoter showed a higher tolerability profile compared to constructs containing the CMV promoter. However, msDNA containing the CMV promoter showed dramatically lower cytokine and liver toxicity responses compared to the CMV precursor parent and the conventional plasmid. See Table 2, below, showing cytokine concentrations (pg/mL) and enzyme concentrations (U/L) of liver function markers at 4 hours and 14 days after injection.
msDNAs were produced from pGL2-SS*-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS* and pGL2-SS*-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA-SS* in an inducible E. coli vector production system using methods described herein and in U.S. Pat. Nos. 9,290,778 and 9,862,954.
Adult wild type mice were administered msDNA-CAG-SecNLuc-2A-eGFP-WPRE-BGpA formulated with a nanocarrier (3 mice) or msDNA-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA formulated with a nanocarrier (3 mice) by three intracerebroventricular (ICV) injections of 1 μg DNA each via implanted cannula on days 0, 14, and 28 after implantation. Animals were euthanized on day 42 after implantation, and sagittal brain sections were collected from the cortex, thalamus, brainstem, and cerebellum.
Comparisons between GFP expression in the cortex, thalamus, and brainstem sections from Mouse #1 of the treatment group injected with msDNA-CAG-SecNLuc-2A-eGFP-WPRE-BGpA and a mouse injected with control plasmid, pcDNA-CMV-5′UTR-SecNLuc-P2A-eGFP-bGHpA, showed that transfection efficiencies and resultant GFP expression were higher with msDNA versus the conventional plasmid (data not shown).
Table 3 below summarizes the transfection efficiencies discussed above for mice injected with msDNA-CAG-SecNLuc-2A-eGFP-WPRE-BGpA (“CAG-WPRE”) or msDNA-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA (“CMV-WPRE”).
Repeated ICV injections via implanted cannula resulted in good overall tissue integrity with no signs of cytotoxicity or neurodegeneration.
The data show that msDNA was redosable and resulted in high transfection efficiencies, biodistribution, and transgene expression in multiple brain regions with no morphological adverse effects.
pcDNA-CMV-5′UTR-SecNLuc-P2A-eGFP-bGHpA (positive control), msDNA-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA, pGL2-SS*-CMV-UTR1-SecNLuc-2A-eGFP-WPRE-BGpA-SS*, msDNA-CAG-SecNLuc-2A-eGFP-WPRE-BGpA, or pGL2-SS*-CAG-SecNLuc-2A-eGFP-WPRE-BGpA-SS* were each lipoplexed with a lipid nanoparticle carrier.
Human T cells (Pan-T(TA+)) and hepatocytes (Huh7) were transfected by 0.3 μg/mL or 1 μg/mL doses of the lipoplexed vectors.
The lipoplexed msDNA vectors showed high expression in both cell types on days 3 and 5 after transfection as compared to the parental and conventional plasmids. See
Lipoplexed msDNA was also well-tolerated in human peripheral blood mononuclear cells (PBMCs) ex vivo. In particular, msDNA showed significantly lower cytokine profile levels in human PBMCs compared to conventional plasmid (data not shown).
Studies were conducted to assess homology directed repair mediated by msDNA as compared to conventional plasmid DNA.
A conventional plasmid was produced with an expression cassette containing a gene of interest (GOI) flanked by 5′ and 3′ homology arms (Plasmid DNA HDR-GOI-HDR).
An msDNA expression vector was produced with the same HDR-GOI-HDR sequence as used in the conventional plasmid flanked by two Super Sequence sites. msDNA containing the HDR-GOI-HDR (msDNA HDR-GOI-HDR) was then produced in an inducible E. coli vector production system using methods described herein and in U.S. Pat. Nos. 9,290,778 and 9,862,954.
Induced pluripotent stem cells (iPSCs) were transfected with equal molarities of either Plasmid DNA HDR-GOI-HDR or msDNA HDR-GOI-HDR along with a CRISPR gene editing system to mediate homology directed repair knock-in (HDR KI) of the GOI.
Homology directed repair knock-in (HDR KI) efficiencies of the GOI was evaluated with fluorescence activated cell sorting (FACS) by counting the total number of integrated healthy iPSCs that expressed the GOI on their surface relative to the total number of transfected cells on days 3, 7, and 15 after transfection. As shown in Q3 of
Expression vectors containing two Super Sequence sites, a CMV enhancer/promoter, an engineered 5′UTR containing an internal minimal intron sequence, and a polygenic expression cassette encoding eGFP and Nluc as described in Examples 1 and 2 were also designed to contain a 3′UTR containing two copies of a human beta-globin polyadenylation signal and 120 adenine nucleotides (i.e., 2huBGpA-A120, SEQ ID NO: 17) and one or more of: (1) a synthetic enhancer (i.e., Enhancer-1 (E1), SEQ ID NO: 12) located at the 5′ end of the CMV enhancer, (2) a WPRE located at the 5′ end of the 3′UTR, (3) a SRF-UCOE located at the 3′ end of the 5′ Super Sequence; and (4) a human CSP-B MAR (huMAR) located at the 3′ end of eGFP. Maps of the designed vectors are shown in
HEK293 cells were separately transfected with (1) a conventional plasmid, pcDNA-CMV-5′UTR-SecNLuc-P2A-eGFP-bGHpA as shown in
On days 2, 3, 7, 10, 14, 21, and 28 after electroporation, luciferase expression was evaluated by measuring the intensity of secreted luciferase from the media of cultured cells as described in Example 2B. See
As shown in
HEK293 cells were transfected with the four expression vectors described in part B of this example. Cells were passaged every 7 days for five passages. At the time of cell passaging, cells were re-seeded at 1/10 of the original cell density. For each cell passage, secreted luciferase expression was measured as described in Example 2B. See
Luciferase expression was detected from cells transfected with any of the msDNA expression vectors at each passage number, showing that the vectors were passed down to daughter cells with durable expression of luciferase.
As shown in
Five synthetic promoter sequences were produced: (1) CAG [E1×3+CBA promoter+intron] (SEQ ID NO: 35), containing three copies of the synthetic enhancer E1 (i.e., 3 copies of SEQ ID NO: 12), a chicken β-actin promoter, and chimeric intron, (2) CAG [E2+CBA promoter+intron] (SEQ ID NO: 36), containing E2 (U100), a chicken β-actin promoter, and chimeric intron, (3) CAG [E1×3+CBA promoter+UTR1] (SEQ ID NO: 37), containing three copies of the synthetic enhancer E1, a chicken β-actin promoter, and 5′UTR1 (i.e., SEQ ID NO: 2), (4) CAG [E2 (U100)+CBA promoter+UTR1] (SEQ ID NO: 38), containing E2 (U100), a chicken β-actin promoter, and 5′UTR1, and (5) CMV enhancer-EF1-UTR1 (SEQ ID NO: 39), containing a CMV enhancer, an EF1a short promoter, and 5′UTR1.
A conventional plasmid was produced containing a CMV enhancer, a chicken β-actin promoter, and chimeric intron and a polygenic expression cassette encoding eGFP and Nluc as described in Examples 1 and 2. A map of the conventional plasmid is shown in
An msDNA expression vector was produced containing two Super Sequences sites, a CMV enhancer, a chicken β-actin promoter, chimeric intron, a polygenic expression cassette encoding eGFP and Nluc, WPRE, and 3′UTR. A map of the vector is shown in
Five msDNA expression vectors were produced containing two Super Sequences sites, a polygenic expression cassette encoding eGFP and Nluc, WPRE, and 3′UTR along with one of synthetic promoters (1)-(5) as described above, with respective vector maps shown in
HEK293 cells were seeded in a 24-well plate at 1×105 cells/well and separately transfected with a complex of lipofectamine and the vectors described in part A of this example at 0.25 pmol DNA/well. Secreted luciferase expression was measured as described in Example 2B at 3 and 6 days after transfection. See
Luciferase expression levels were higher for all msDNA expression vectors as compared to the conventional plasmid. The highest expression was observed with 4-6-pGL2-SS*-CMV enhancer-EF1-UTR1-SecNLuc-2A-eGFP-WPRE-3′UTR(108 to 120 polyA (4-6: CMV-EF1-UTR1-W-3′UTR), which contains the EF-1 promoter element in combination with the CMV enhancer and 5′UTR1.
The impact of modifications to the Super Sequence (SS) and the expression cassettes of the expression vectors as described in the present disclosure will be evaluated in terms of transfection efficiencies, expression of nucleic acid sequences of interest (including reporter genes, such as polygenic GFP and luciferase expression cassettes as described in Examples 1 and 2), and durability/expansion of the vectors in dividing cells (including rapid and slow dividing cells). Modifications to the SS also will be evaluated for restriction enzyme activity on these sites.
Modifications will include individual modifications and combinations such as, but not limited to, an endonuclease target sequence integrated in non-binding regions for the recombinases in the SS between the vector backbone and the cleavage sites for the recombinases, a CAG promoter integrated between the 3′ end of the first target sequence for the first recombinase (i.e., the 3′ end of the 5′ SS) and 5′ to the promoter in the expression cassette, a CMV enhancer integrated between the 3′ end of the first target sequence for the first recombinase (i.e., the 3′ end of the 5′ SS) and 5′ to the promoter in the expression cassette, an Enhancer-1 sequence located 5′ to a CMV enhancer and/or 3′ to a UCOE, a CMV, EF1, SV40, CAG, Rho, VDM2, HCR, or HLP promoter or variant thereof, a CMV promoter variant, an EF1-alpha promoter, a synthetic promoter, a 5′UTR comprising an intron integrated in the expression cassette between a promoter and nucleic acid sequence of interest with or without non-coding sequences integrated within the intron (e.g., a 5′UTR comprising the nucleic acid sequence of any one of SEQ ID NOs:2-5), a vertebrate chromatin insulator integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal, a woodchuck hepatitis virus post-transcriptional regulatory element integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal, a scaffold/matrix attachment region integrated in the expression cassette between the nucleic acid of interest and a polyadenylation signal, an ubiquitous chromatin opening element located 5′ to the promoter in the expression cassette (e.g., at the 3′ of the 5′ SS and prior to other sequences in the expression cassette), a 3′UTR integrated in the expression cassette between the nucleic acid of interest and the 3′ SS, such as directly following a stop codon (e.g., a 3′UTR comprising the nucleic acid sequence of any one of SEQ ID NOs: 13-16), and/or a poly(A) tail (e.g., as the 3′ end of a 3′UTR) comprising 100 to 120 adenine nucleotides.
The disclosure is not to be limited in scope by the specific aspects described herein. Indeed, various modifications of the disclosure in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
Other aspects are within the following claims.
This application is a continuation of PCT Application No. PCT/IB2022/055620, filed Jun. 16, 2022, which claims the priority benefit of U.S. Provisional Application Nos. 63/211,343, filed Jun. 16, 2021, 63/306,015, filed Feb. 2, 2022, and 63/331,638, filed Apr. 15, 2022, which are incorporated herein by reference in their entireties.
Number | Date | Country | |
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63211343 | Jun 2021 | US | |
63306015 | Feb 2022 | US | |
63331638 | Apr 2022 | US |
Number | Date | Country | |
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Parent | PCT/IB2022/055620 | Jun 2022 | WO |
Child | 18541459 | US |