Patent Application
20220267802
The delivery of nucleic acids to cells finds many important applications in human health, biochemical production, and scientific discovery. Some of the most commonly vectors used for gene delivery include lentivirus (LV), retrovirus (RV), herpes simplex virus-1 (HSV-1) and adeno-associated virus (AAV). Nonetheless, the use of vectors for delivering nucleic acids are limited in size capacity. This limitation prevents delivery of large genes or other large nucleic acid sequences that are necessary for treatment of diseases and other gene delivery applications.
Provided herein is a technology for co-delivering to a cell (e.g., in vivo or ex vivo) enzymes capable of rearranging nucleic acid, such as site-specific recombinases, to directly assemble (e.g., covalently join) nucleic acid segments of, for example, a gene of interest. These enzymes can be programmed to join multiple nucleic acid molecules (e.g., segments) together efficiently in a site-directed and order-specific manner, resulting, for example, in expression of a full length protein encoded by the nucleic acid segments, following a single translation event, without the need for protein engineering. Moreover, site-specific recombinases do not rely heavily on cellular components and machinery, providing a more consistent and tunable assembly strategy across cell types, relative to current strategies that use pre-existing repair machinery encoded in the target cells, which has proven to be inefficient, variable between cell type, and difficult to control.
In some embodiments, the enzyme capable of rearranging nucleic acid is a site-specific recombinase (SSR), which is a small enzyme (e.g., ˜200 to ˜700 amino acids) that catalyzes the transfer and rearrangement of nucleic acids by executing nucleic acid-binding, cutting, transfers and ligation reactions. SSRs carry out these activities on a unique sequence referred to as a recombination site (RS), which is typically between 27 to 250 base-pairs in sequence length. Depending on the placement and orientation of the RS sequences, SSRs can invert, delete, or translocate nucleic acids. SSRs can be classified based on which amino acid residue is primarily responsible for covalent attachment to nucleic acids: tyrosine (tyrosine recombinases) or serine (serine recombinases) residues.
Adeno-associated virus (AAV) vectors have been included in virus-based products federally-approved in the U.S. for in vivo gene therapy of inherited diseases, with many more currently undergoing in clinical trials. Despite much interest around AAV as safe and effective vehicle for gene delivery, AAV cannot package sequences longer than the 4.7 kilobases (kb). More than 4% of the human genes are longer than 4.7 kb, while 11.8% exceed 3 kb (2398 total genes). Thus, in some embodiments, AAV vectors are used to deliver nucleic acid molecules to a cell.
Some aspects of the present disclosure provide a method comprising delivering to a cell (a) a first vector comprising a first segment of a nucleic acid segment and a first recombination site, (b) a second vector comprising a second segment of the nucleic acid and a second recombination site, (c) and a cognate site-specific enzyme or a nucleic acid encoding a cognate site-specific nucleic acid-rearranging enzyme that catalyzes a recombination event to join the first segment to the second segment, thereby forming a transcription product.
In some embodiments, (c) comprises the nucleic acid encoding a cognate site-specific nucleic acid-rearranging enzyme that catalyzes joining of the first segment to the second segment.
In some embodiments, the method further comprises at least one additional vector comprising at least one addition segment of the nucleic acid and at least one addition recombination site.
In some embodiments, the first vector or second vector comprises the nucleic acid encoding the cognate site-specific nucleic acid-rearranging enzyme.
In some embodiments, a third vector comprises nucleic acid encoding the cognate site-specific nucleic acid-rearranging enzyme.
In some embodiments, the first vector comprises a promoter operably linked to the first segment of the nucleic acid. In some embodiments, the third vector comprises a promoter operably linked to the nucleic acid encoding the cognate site-specific nucleic acid-rearranging enzyme.
In some embodiments, the second vector comprise a post-transcriptional regulator element (e.g., woodchuck hepatitis virus post-transcriptional regulator element (WPRE)). In some embodiments, the third vector comprise a post-transcriptional regulator element (e.g., WPRE).
In some embodiments, following the transcription event the transcription product comprises a scar recombination site located between the first segment and the second segment.
In some embodiments, the first vector further comprises a splice donor site and the second vector comprises a branch point site and a splice acceptor site, and following a recombination event, the scar recombination site of the transcription product is flanked by (i) the splice donor site and (ii) the branch point site and the splice acceptor site.
In some embodiments, the first segment, second segment, and/or at least one additional segment are exons of a gene of interest.
In some embodiments, the gene of interest is a therapeutic gene, optionally selected from the group consisting of any of the therapeutic genes listed in Table 1.
In some embodiments, the gene of interest encodes a gene-editing protein, optionally a Cas9 enzyme or a Cas9 enzyme variant (e.g., Cas9 fused to a transcriptional activator, a transcriptional repressor, or a deaminase).
In some embodiments, the first vector, the second vector, and/or the at least one additional vector is selected from the group consisting of lentiviral vectors, retroviral vectors, adenoviral vectors, and adeno-associated viral vectors. In some embodiments, the first vector, the second vector, and/or the at least one additional vector is an adeno-associated viral vector.
In some embodiments, the site-specific enzyme is selected from the group consisting of site-specific recombinases, DDE transposases, DDE LTR-retrotransposases, and target-primed retrotransposases.
In some embodiments, the site-specific enzyme is a site-specific recombinase (SSR) selected from the group consisting of serine recombinases, RKHRY-type recombinases, and HUH-type recombinase.
In some embodiments, the SSR is a serine recombinase selected from the group consisting of small serine recombinases, large serine integrases, and IS607-like serine transposases.
In some embodiments, the serine recombinase is a small serine recombinase selected from the group consisting of resolvases, invertases, and resolvase-invertases. In some embodiments, the small serine recombinase is a resolvase selected from the group consisting of Tn3 resolvase and gamma-delta resolvase. In some embodiments, the small serine recombinase is an invertase selected from the group consisting of Gin invertase and Hin invertase. In some embodiments, the small serine recombinase is a resolvase-invertase selected from the group consisting of BinT resolvase-invertase and beta resolvase-invertase.
In some embodiments, the serine recombinase is a large serine recombinase selected from the group consisting of Bxb1 recombinase, TP901-1 recombinase, PhiC31 recombinase, TG1 recombinase, and PhiRv1 recombinase. In some embodiments, the SSR is Bxb1 recombinase.
In some embodiments, the SSR is a RKHRY-type recombinase selected from the group consisting of tyrosine recombinases, tyrosine integrases, tyrosine invertases, tyrosine shufflons, tyrosine transposases, topoisomerase IB, and telomere resolvases.
In some embodiments, the RKHRY-type recombinase is a tyrosine recombinase selected from the group consisting of Cre recombinase, Flp recombinase, XerC/D recombinase, and XerA recombinase. In some embodiments, the RKHRY-type recombinase is a tyrosine integrase selected from the group consisting of Lambda integrase, P2 integrase, and HK022 integrase. In some embodiments, the RKHRY-type recombinase is a tyrosine invertase selected from the group consisting of FimB invertase, FimE invertase, and HbiF invertase. In some embodiments, the RKHRY-type recombinase is a tyrosine Rci shufflon. In some embodiments, the RKHRY-type recombinase is a tyrosine transposase selected from the group consisting of crypton transposases, DIR transposases, Ngaro transposases, PAT transposases, Tec transposases, Tn916 transposases, and CTnDOT transposases.
In some embodiments, the SSR is a HUH-type recombinase selected from the group consisting of Y1-transposases of IS200/IS605 (e.g., IS608 TnpA and ISDra2), and ISC transposases (e.g., IscA), helitron transposases, IS91 transposases, AAV Rep78 transposases, and TrwC relaxases.
In some embodiments, the site-specific enzyme is a DDE transposase selected from the group consisting of Tc1/mariner transposases, piggyBac transposases, Transib transposases, hAT transposases, Tn5 transposases, P elements, mutator transposases, and CMC transposases.
In some embodiments, the site-specific enzyme is a DDE LTR-retrotransposase selected from the group consisting of Ty3/gypsy and HIV integrase.
In some embodiments, the site-specific enzyme is a target-primed retrotransposase selected from the group consisting of LINE-1 and Group II introns.
In some embodiments, the first vector, second vector, third vector, and/or site-specific nucleic acid-rearranging enzyme are delivered to the cell via electroporation, polymer formulation, or other transfection reagent.
Other aspects of the present disclose provide methods that comprise delivering to a cell at least two viral vectors, each comprising a payload, using a site-specific recombinase. In some embodiments, the viral vectors are adeno-associated viral vectors. In some embodiments, the site-specific recombinase is Bxb1 recombinase.
Further aspects of the present disclose provide a cell comprising the first vector, the second vector, and the cognate site-specific enzyme or the nucleic acid encoding the cognate site-specific nucleic acid-rearranging enzyme of any one of the preceding claims. In some embodiments, the cell is a mammalian cell, optionally a human cell.
Still other aspects of the present disclose provide a composition comprising the first vector, the second vector, and the cognate site-specific enzyme or the nucleic acid encoding the cognate site-specific nucleic acid-rearranging enzyme of any one of the preceding claims and at least one additional reagent (e.g., cell culture media or buffer).
Yet other aspects of the present disclose provide a kit comprising the first vector, the second vector, and the cognate site-specific enzyme or the nucleic acid encoding the cognate site-specific nucleic acid-rearranging enzyme of any one of the preceding claims and at least one additional reagent (e.g., cell culture media or buffer), wherein the first segment, the second segment, and/or the at least one additional segment are replaced by a multiple cloning site.
Also provided herein is a vector comprising any one of the vector designs of
Yet other aspects herein provide a kit comprising vectors that comprise the 3-vector design or the 2-vector design of
Further aspects of the present disclosure provide a nucleic acid vector comprising, in a 5′ to 3′ orientation, a coding region, a splice donor site, a recombination site, and optionally a 5′ LTR and a 3′ LTR. In some embodiments, the vector further comprises a promoter upstream from and operably linked to the coding region, and optionally further comprising 5′ LTR and a 3′ LTR. In some embodiments, the vector further comprises a recombination site upstream from the coding region. Yet other aspects provide a nucleic acid vector comprising, in a 5′ to 3′ orientation, a recombination site, a splice acceptor site, a coding region, optionally a post-transcriptional regulator element, and optionally a 5′ LTR and a 3′ LTR. In some embodiments, the vector further comprises a promoter, a recombination site, a coding region that encodes a site-specific nucleic acid-rearranging enzyme (e.g., as site-specific recombinase), and optionally a post-transcriptional regulator element, wherein the promoter is operably linked to the coding region that encodes a site-specific nucleic acid-rearranging enzyme. Still other aspects provide a nucleic acid vector comprising, in a 5′ to 3′ orientation, a promoter operably linked to a coding region that encodes a site-specific nucleic acid-rearranging enzyme (e.g., as site-specific recombinase), a post-transcriptional regulator element, optionally a 5′ LTR and a 3′ LTR, and optionally a recombination site upstream from the coding region and another recombination site downstream from the coding region.
Some aspects of the present disclosure provide method comprising delivering to a cell (a) a first vector comprising a first segment of a gene of interest and a first recombination site, (b) a second vector comprising a second segment of the gene of interest and a second recombination site, (c) and a cognate site-specific recombinase or a nucleic acid encoding a cognate site-specific recombinase. In some embodiments, (c) is a nucleic acid encoding a cognate site-specific recombinase.
In some embodiments, the nucleic acid encoding a cognate site-specific recombinase is delivered on the first or second vector. In other embodiments, the nucleic acid encoding a cognate site-specific recombinase is delivered on a third vector.
Other aspects of the present disclosure provide a method comprising delivering to a cell (a) a first vector comprising a first nucleic acid comprising, optionally in a 5′ to 3′ orientation, a first promoter operably linked to a first segment of a gene of interest, a splice donor site, and a first recombination site, wherein the first nucleic acid is flanked by a first pair inverted terminal repeat sequences (ITRs)/long terminal repeats (LTRs), (b) a second vector comprising a second nucleic acid comprising, optionally in a 5′ to 3′ orientation, a second recombination site, a splice acceptor site, a second segment of the gene of interest, and a post-transcriptional regulator element, optionally WPRE, wherein the second nucleic acid is flanked by a second pair of ITR/LTR sequences, and (c) a third vector comprising a third nucleic acid comprising a second promoter operably linked to a nucleotide sequence encoding a cognate site-specific recombinase and a post-transcriptional regulator element, optionally WPRE, wherein the third nucleic acid is flanked by a second pair of ITR/LTR sequences.
In some embodiments, the cognate site-specific recombinase catalyzes a recombination event to join the first segment to the second segment.
In some embodiments, the vector is a plasmid.
In some embodiments, the vector is a viral vector. In some embodiments, wherein the viral vector is selected from the group consisting of adeno-associated viral vectors, adenoviral vectors, lentiviral vectors, and retroviral vectors. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector, optionally an AAV2 vector.
In some embodiments, the site-specific recombinase is a serine recombinase. In some embodiments, the serine recombinase is selected from the group consisting of Bxb1 recombinase, TP901-1 recombinase, PhiC31 recombinase, TG1 recombinase, and PhiRv1 recombinase. In some embodiments, the serine recombinase is a Bxb1 recombinase.
In some embodiments, the site-specific recombinase is a tyrosine recombinase. In some embodiments, the tyrosine recombinase is selected from the group consisting of Cre recombinase, Flp recombinase, XerC/D recombinase, and XerA recombinase. In some embodiments, the tyrosine recombinase is Cre recombinase.
In some embodiments, the first segment is a first exon of the gene of interest, and the second segment is a second exon of the gene of interest. In some embodiments, the gene of interest is a therapeutic gene of interest and/or encodes a therapeutic protein. In some embodiments, the gene of interest encodes a Cas protein, optionally a Cas9 or Cas12a protein, optionally fused to a transcriptional activator, a transcriptional repressor, or a deaminase.
Also provided herein, in some aspects, is a composition, cell, or kit comprising (a) a first vector comprising a first segment of a gene of interest and a first recombination site, (b) a second vector comprising a second segment of the gene of interest and a second recombination site, (c) and a cognate site-specific recombinase or a nucleic acid encoding a cognate site-specific recombinase.
Further provided herein, in some aspects, is a composition, cell, or kit comprising (a) a first vector comprising a first nucleic acid comprising, optionally in a 5′ to 3′ orientation, a first promoter operably linked to a first segment of a gene of interest, a splice donor site, and a first recombination site, wherein the first nucleic acid is flanked by a first pair ITR/LTR sequences, (b) a second vector comprising a second nucleic acid comprising, optionally in a 5′ to 3′ orientation, a second recombination site, a splice acceptor site, a second segment of the gene of interest, and a post-transcriptional regulator element, optionally WPRE, wherein the second nucleic acid is flanked by a second pair of ITR/LTR sequences, and (c) a third vector comprising a third nucleic acid comprising a second promoter operably linked to a nucleotide sequence encoding a cognate site-specific recombinase and a post-transcriptional regulator element, optionally WPRE, wherein the third nucleic acid is flanked by a second pair of ITR/LTR sequences.
A vector used as provided herein, in some embodiments, is a viral vector. In some embodiments, a viral vector is not a naturally occurring viral vector. The viral vector may be from adeno-associated virus (AAV), adenovirus, herpes simplex virus, lentiviral, retrovirus, varicella, variola virus, hepatitis B, cytomegalovirus, JC polyomavirus, BK polyomavirus, monkeypox virus, Herpes Zoster, Epstein-Barr virus, human herpes virus 7, Kaposi's sarcoma-associated herpesvirus, or human parvovirus B 19. Other viral vectors are encompassed by the present disclosure.
In some embodiments, a viral vector is an AAV vector. AAV is a small, non-enveloped virus that packages a single-stranded linear DNA genome that is approximately 5 kb long and has been adapted for use as a gene transfer vehicle (Samulski, R J et al., Annu Rev Virol. 2014; 1(1):427-51). The coding regions of AAV are flanked by inverted terminal repeats (ITRs), which act as the origins for DNA replication and serve as the primary packaging signal (McLaughlin, S K et al. Virol. 1988; 62(6): 1963-73; Hauswirth, W W et al. 1977; 78(2):488-99). Thus, an AAV vector typically includes ITR sequences. Both positive and negative strands are packaged into virions equally well and capable of infection (Zhong, L et al. Mol Ther. 2008; 16(2):290-5; Zhou, X et al. Mol Ther. 2008; 16(3):494-9; Samulski, R J et al. Virol. 1987; 61(10):3096-101). In addition, a small deletion in one of the two ITRs allows packaging of self-complementary vectors, in which the genome self-anneals after viral uncoating. This results in more efficient transduction of cells but reduces the coding capacity by half (McCarty, D M et al. Mol Ther. 2008; 16(10): 1648-56; McCarty, D M et al. Gene Ther. 2001; 8(16): 1248-54).
In some embodiments, a vector comprises a nucleotide sequence encoding a nucleic acid sequence operably linked to a promoter (promoter sequence). In some embodiments, the promoter is an inducible promoter (e.g., comprising a tetracycline-regulated sequence). Inducible promoters enable, for example, temporal and/or spatial control of gene expression.
A promoter control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. A promoter may also contain sub-regions at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. Promoters may be constitutive, inducible, activatable, repressible, tissue-specific or any combination thereof. A promoter drives expression or drives transcription of the nucleic acid sequence that it regulates. Herein, a promoter is considered to be operably linked when it is in a correct functional location and orientation in relation to a nucleic acid sequence it regulates to control (“drive”) transcriptional initiation and/or expression of that sequence.
An inducible promoter is one that is characterized by initiating or enhancing transcriptional activity when in the presence of, influenced by or contacted by an inducing agent. An inducing agent may be endogenous or a normally exogenous condition, compound or protein that contacts an engineered nucleic acid in such a way as to be active in inducing transcriptional activity from the inducible promoter.
Inducible promoters for use in accordance with the present disclosure include any inducible promoter described herein or known to one of ordinary skill in the art. Examples of inducible promoters include, without limitation, chemically/biochemically-regulated and physically-regulated promoters such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid/retinoid/thyroid 25 receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature/heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light responsive promoters from plant cells).
The vectors of the present disclosure may be generated using standard molecular cloning methods (see, e.g., Current Protocols in Molecular Biology, Ausubel, F. M., et al., New York: John Wiley & Sons, 2006; Molecular Cloning: A Laboratory Manual, Green, M. R. and Sambrook J., New York: Cold Spring Harbor Laboratory Press, 2012; Gibson, D. G., et al., Nature Methods 6(5):343-345 (2009), the teachings of which relating to molecular cloning are herein incorporated by reference).
The methods and compositions of the present disclosure may be used, for example, to deliver to a cell a payload. A payload, herein, can be any polynucleotide (nucleic acid) of interest. In some embodiments, a payload is a nucleic acid that encodes a molecule of interest or a portion of a molecule of interest, such as, for example, a polypeptide (e.g., protein) of interest. Thus, in some embodiments, a payload is a gene of interest or a segment of a gene of interest.
Vectors described herein are limited in size capacity, which prevents delivery of large nucleic acid sequences. Thus, these large nucleic acid sequences may be divided among two or more vectors, delivered to a cell, and then assembled within the cell. As described above, AAV, for example, has a capacity of only 4.7 kb. AAV vectors may be used as described herein to deliver nucleic acids that are larger than 4.7 kb by dividing the nucleic acid into two or more segments, each segment having a size of smaller than 4.7 kb. Each segment can be delivered to a cell on an independent AAV vector. Other viral vectors may be used in a similar manner, dividing the nucleic acid into segments, guided by size capacity of the vector. Thus, a single gene, for example, may be delivered to a cell by delivering multiple vectors, each payload of the vector being a segment of the gene.
In some embodiments, the methods and compositions of the present disclosure are used to deliver a therapeutic gene to a cell. For example, a first second and a second segment described herein may together (when joined and transcribed/translated together) form a therapeutic gene or encode a therapeutic protein. Table 1 provides examples of therapeutic genes/proteins and their related diseases.
The size of the therapeutic gene, other gene of interest, or other nucleic acid of interest may vary. In some embodiments, the nucleic acid (e.g., gene) has a size of at least 4 kilobases (kb). For example, the gene may have a size of at least 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 kb. In some embodiments, the nucleic acid (e.g., therapeutic gene or other gene of interest) has a size of 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, or 4-5 kb. In some embodiments, the nucleic acid (e.g., therapeutic gene or other gene of interest) has a size of 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, or 5-6 kb. In some embodiments, the nucleic acid (e.g., therapeutic gene or other gene of interest) has a size of 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, or 6-7 kb. In some embodiments, the nucleic acid (e.g., therapeutic gene or other gene of interest) has a size of 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, or 7-8 kb. In some embodiments, the nucleic acid (e.g., therapeutic gene or other gene of interest) has a size of 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, or 8-9 kb. In some embodiments, the nucleic acid (e.g., therapeutic gene or other gene of interest) has a size of 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, or 9-10 kb. In some embodiments, the nucleic acid (e.g., therapeutic gene or other gene of interest) has a size of 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, or 10-11 kb.
The size of a nucleic acid segment forming part of a gene or encoding part of a protein may vary. Any of the nucleic acid segments (e.g., a first segment and/or a second segment) may have a size of 0.5 kb to 10 kb. Larger segments are also contemplated herein. In some embodiments, a first and/or second segment has a size of 0.5 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, or 10 kb. In some embodiments, a first and/or second segment has a size of 1-10 kb, 2-10 kb, 3-10 kb, 4-10 kb, 5-10 kb, 6-10 kb, 7-10 kb, 8-10 kb, or 9-10 kb.
In some embodiments, the methods and compositions of the present disclosure are used to deliver nucleic acid molecules that collectively encode a protein (e.g., enzyme) used in gene editing. For example, the methods and compositions of the present disclosure may be used to deliver nucleic acid molecules that collectively encode Cas9 protein (or another Cas protein, such as Cas12a protein) and/or guide RNA (gRNA). Cas9 protein is from Streptococcus pyogenes and is a 1367 amino acid (4.101 kb) RNA-guided DNA endonuclease that has been adopted for making DNA edits in genomes of living human cells. Other examples include larger Cas9 variations which have been fused with additional sequences, such as transcription activators (e.g. VP64, p65), transcription repressors (e.g., KRAB), and deaminases for further functionality; these additional sequences further complicate and prevent the packaging into a single AAV vector, for example.
A site-specific nucleic acid-rearranging enzyme is any enzyme that can catalyze the reciprocal exchange of nucleic acid between define sites, referred to herein as recombination sites.
In some embodiments, the site-specific enzyme is selected from the group consisting of site-specific recombinases, transposases, and retrotransposases.
In some embodiments, the site-specific enzyme is a site-specific recombinase. Site-specific recombinases (SSRs) can rearrange nucleic acid (e.g., DNA) segments by recognizing and binding to short nucleic acid sequences (recombination sites), at which they cleave the nucleic acid backbone, exchange the two nucleic acids (e.g., DNA helices) involved and rejoin the nucleic acid strands. Based on amino acid sequence homology and mechanistic relatedness, most site-specific recombinases are grouped into one of two families: the tyrosine recombinase family or the serine recombinase family. The names stem from the conserved nucleophilic amino acid residue that they use to attack the DNA and which becomes covalently linked to it during strand exchange. Non-limiting examples of site-specific recombinases are described herein and include, Flp, KD, B2, B3, R, Cre, VCre, SCre, Vika, Dre, λ-Int, HK022, φC31, Bxb1, Gin, and Tn3. Table 2 provides non-limiting examples of site-specific recombinases and their corresponding recombination sites.
S. cerevisiae
K.
drosophilarum
Z. bailii
Z. bisporus
Z. rouxii
Vibrio sp.
Shewattella
V.
coralliilyticus
E. coli
Non-limiting examples of tyrosine recombinase family molecules that may be used as a site-specific recombinase include Cre, Flp, XerC/D, XerA, Lambda, P2, HK022, FimB, FimE, HbiF, Rci, Cryptons, DIRS, Ngaro, PAT, Tec, Tn916, CTnDOT, topoisomerase IB, telomere resolvases, Y1-transposases of IS200/IS605 (e.g., IS608 TnpA, ISDra2), ISC (e.g. IscA), Helitrons, IS91, AAV Rep78, TrwC relaxase, MrpA, XerH, XerS, DAI, SSV, PhiCh1, pNOB, pTN3, IntC, IntG, IntI, and SNJ2 recombinases.
Non-limiting examples of serine recombinase family molecules that may be used as a site-specific recombinase include Tn3, gamma-delta, Gin, Hin, Gin, Hin, Bxb1, TP901-1, PhiC31, TG1, PhiRv1, and C.IS607-like serine transposase.
Other site-specific recombinases may be used. For example, Yang L et al. provides phage integrases that may be used in accordance with the present disclosure (see, e.g., Supplementary Table 1 of Yang Let al. Nat Methods. 2014; 11(12): 1261-1266, incorporated herein by reference). Table 3 below provides additional examples of site-specific recombinases that may be used as provided herein.
In some embodiments, a recombination site is positioned between a promoter and a coding region for a site-specific recombinase, which results in promoter cleavage after one recombination event, thus preventing uncontrolled expression of the site-specific recombinase. The design of this “protective” switch can be used to address any off-target genome effects due to potential high copy number expression and prolonged exposure of the site-specific recombinase.
In some embodiments, the site-specific enzyme is transposase. A transposase is an enzyme that binds to the end of a transposon and catalyzes its movement to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism. Most transposases include a DDE motif (herein referred to as DDS transposases), which is the active site that catalyzes the movement of the transposon. Aspartate-97, Aspartate-188, and Glutamate-326 make up the active site, which is a triad of acidic residues.
In some embodiments, the site-specific enzyme is a retrotransposase. Retrotransposons are genetic elements that can amplify themselves in a genome and are ubiquitous components of the DNA of many eukaryotic organisms. These DNA sequences are first transcribed into RNA, then converted back into identical DNA sequences using reverse transcription, and these sequences are then inserted into the genome at target sites. In some embodiments, the retrotransposase is a long-terminal repeat (LTR) transposase. LTR retrotransposons have direct LTRs that range from ˜100 bp to over 5 kb in size. LTR retrotransposons are further sub-classified into the Ty1-copia-like (Pseudoviridae), Ty3-gypsy-like (Metaviridae), and BEL-Pao-like groups based on both their degree of sequence similarity and the order of encoded gene products. In some embodiments, the retrotransposase comprises a DDE motif and a LTR (referred to herein as a DDE LTR-retrotransposase). In some embodiments, the retrotransposase is a target-primed retrotransposases, such as a long interspersed nuclear element (LINE). retrotransposase.
The methods herein may be used to deliver payloads to any cell. In some embodiments, the cell is a cell of a model organism, such as mouse, rat, or monkey. In some embodiments, the cell is a mammalian cell. The mammalian cell may be, for example, a human cell.
First, nucleic acid vectors are generated. Each vector that is delivered and assembled together contains a recombination site (RS) sequence of the specific site-specific recombinase (SSR) that is used. Long genes that cannot be contained in a single vector are designed into multiple nucleic acid segments to be split among multiple vectors (
The methods described herein have been demonstrated in living human embryonic kidney (HEK293T) cells. Sanger sequencing confirmed joining of two AAV2 vectors by Bxb1 integrase using a 3-vector design strategy (
Flow cytometric results showed expression of assembled mKate fluorescent protein gene from two AAV2 vectors by Bxb1 integrase using a 2-vector design strategy (
Cre-mediated assembly of two DNA fragments was tested in vitro. Two double-stranded DNA fragments containing lox sites were created by PCR using fluorescently labelled primers (Cy5 or IRD800) (
The assembly of plasmid DNA by Cre recombinase was tested in living mammalian cells. As shown in
Flow cytometry was performed on the cells 48 hours post-transfection and GFP mean fluorescence intensity (MFI) was determined on single cells containing BFP fluorescence. As shown in
Plasmid DNA was isolated and PCR was performed using primer sites indicated in
magadii]
gibbonsii]
nitratireducens]
amylolytica]
acetivorans]
rufus]
mazei]
daqingensis]
rubra]
gerundensis]
freundii]
protegens Pf-5]
gladioli BSR3]
baumannii
oralis]
pfennigii]
corrodens]
gonorrhoeae]
cerevisiae]
fermentati]
bailii]
blattae CBS 6284]
eubayanus]
pseudotuberculosis]
influenzae]
enterocolitica]
dianthicola]
mallotivora]
radicincitans]
marcescens]
vestfoldensis
cenocepacia PC184]
mobile]
aestuarii]
aquimarina]
pneumoniae]
sonnei]
baumannii AB0057]
pneumoniae]
freundii]
serovar
Typhimurium]
jeotgali]
rufus]
tokodaii]
hospitalis]
islandicus]
distributa]
solfataricus
islandicus]
veneficus]
kodakarensis]
ferrophilus]
kodakarensis]
acetivorans]
prieurii
nautili]
sonnei]
enterica
serovar
Typhimurium]
coli]
enterica]
enterica]
enterica]
braakii]
aceae]
marismortui
utahensis
mukohataei
volcanii
larsenii
utahensis
pharaonis
pharaonis
thermotolerans
vallis mortis]
Mexican
fusellovirus
ovoid virus 1]
solfataricus]
sedula]
acidophilum]
furiosus]
kodakarensis]
arvoryzae]
Boonei]
nautili]
abyssi]
baltica]
oleovorans]
luteus]
mccartyi]
aerogenes]
camosus]
solani]
ferrireducens]
fontium]
wolfei]
coli
coli
mirabilis]
limicola]
coelicolor]
1Hacein-Bey-Abina, S., et al. (2008). “Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1.” J Clin Invest 118(9): 3132-3142.
3Merrick, C. A., et al. (2016). “Rapid Optimization of Engineered Metabolic Pathways with Serine Integrase Recombinational Assembly (SIRA).” Methods Enzymol 575: 285-317.
All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.
The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value.
Where a range of values is provided, each value between the upper and lower ends of the range are specifically contemplated and described herein.
This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application No. 62/874,241 filed on Jul. 15, 2019, which is incorporated by reference herein in its entirety.
This invention was made with government support under DE-FG02-02ER63445 awarded by the Department of Energy. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US20/41950 | 7/14/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62874241 | Jul 2019 | US |
