CN113355354A - System and method for controlling cell behavior - Google Patents
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- CN113355354A CN113355354A CN202110246143.0A CN202110246143A CN113355354A CN 113355354 A CN113355354 A CN 113355354A CN 202110246143 A CN202110246143 A CN 202110246143A CN 113355354 A CN113355354 A CN 113355354A
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Abstract
The generation of antigen-specific immune cells (e.g., T cells) by controlled ex vivo induction or expansion can provide highly specific and beneficial T cell therapies. The present disclosure provides T cell manufacturing methods and therapeutic T cell compositions, e.g., for treating subjects with cancer and other conditions, diseases, and disorders, as well as individual antigen-specific T cell therapies.
Description
Cross-referencing
The present application claims priority from international patent application PCT/CN2020/077533 filed on 3/2020/077545 in 2020, international patent application PCT/CN2020/077545 on 3/2020, international patent application PCT/CN2020/077535 on 3/2020, international patent application PCT/CN2020/077547 on 3/2020, international patent application PCT/CN2020/077536 on 3/2020, and international patent application PCT/CN2020/077557 on 3/2020, each of which is filed on 3/2020, is hereby incorporated by reference in its entirety.
Background
Gene editing techniques have evolved rapidly over the past few decades. Gene editing can be used in a variety of applications, including scientific research, medical diagnostics, and therapeutic development. Gene editing may involve one or more manipulations of a gene, for example, the addition, deletion, or substitution of a target gene or portion thereof at a single or multiple loci within a genome. This may allow therapeutically beneficial alterations to be introduced into the genome of the cell, resulting in an engineered cell, for example, by replacing a mutated or defective gene encoding a non-functional protein with a gene having the correct sequence encoding a functional protein.
However, gene editing techniques still suffer from some drawbacks. For example, genetic manipulation of cells (e.g., isolated cells) can be associated with toxicity and premature cell death, and can result in inefficient recovery of the manipulated cells. In some cases, multiple sequential gene manipulations may be required and may accelerate or even exacerbate the toxic effects of gene manipulations. Genetic editing of primary cells, terminally differentiated cells with limited proliferative capacity can create other technical challenges due to the limited time window for genetic manipulation and the limited ability to recover enough cells for appropriate downstream applications.
Disclosure of Invention
In view of the above, attempts to increase cell life by immortalization may be a reasonable approach to solve this problem. At the same time, however, one skilled in the art may be aware that immortalized cells may not be suitable for certain applications, such as in vivo use. Furthermore, cell immortalization can often resort to the introduction of viral genes into cells, which can pose safety concerns. The present disclosure addresses at least the above-described shortcomings of gene editing by providing systems and methods, for example, for modulating cell immortalization. In one aspect, cells can be suitably manipulated to exhibit an extended lifespan and enhanced proliferative capacity (i.e., immortalization) such that the cells can undergo two or more gene editing events while retaining their intrinsic cellular properties. Using the methods and compositions disclosed herein, cell immortalization can circumvent accidental tumorigenic transformation of cells. The present disclosure also provides systems and methods for inactivating induced cell immortalization by introducing one or more gene edits of interest (e.g., genetic manipulations for improving allogeneic (allogenic) or xenogeneic (xenogeneic) compatibility of the cells). The subject systems and methods of the present disclosure are useful for a number of biological applications, including but not limited to engineered immune cell therapy and the preparation of genetically modified animals for xenotransplantation.
In one aspect, the present disclosure describes a system for modulating cell immortalization comprising: a first polynucleotide sequence comprising an immortalizing gene, wherein expression of said immortalizing gene results in the immortalization of a cell; and a second polynucleotide sequence comprising a replacement gene replacing the immortalized gene or a fragment thereof, wherein expression of the immortalized gene in the cell is reduced upon induction of the system with a modulator that affects the exchange between the immortalized gene and the replacement gene.
In some embodiments, the first polynucleotide sequence is chromosomal.
In some embodiments, the first polynucleotide sequence is integrated at one or more sites in the genome of the cell, wherein the one or more sites are selected from the group consisting of: AAVS1 site, GGTA1 site, CMAH site, B4GALNT2 site, B2M site, ROSA26 site, COLA1 site and TIGRE site.
In some embodiments, the first polynucleotide sequence is integrated into the genome of the cell by a nucleic acid editing moiety comprising a nucleic acid cleavage moiety.
In some embodiments of any of the systems disclosed above, the replacement gene is capable of enhancing allogeneic or xenogeneic compatibility of the cell.
In some embodiments, the replacement gene encodes a human copy selected from the group consisting of: B2M, HLAE, CD47, PDL1, CD46, CD55, CD59, A20, THBD, TFPI, CD39, EPCR, FASL, CIITA, CTLA-Ig and HO-1.
In some embodiments of any of the systems disclosed above, the cell is incapable of expressing a functional copy of an incompatible allogeneic or xenogeneic epitope.
In some embodiments, the incompatible allogeneic or xenogeneic epitope is selected from: GGTA, CMAH, and B4 Gal.
In some embodiments of any one of the systems disclosed above, the cell is a non-human mammalian cell. In some embodiments, the cell is a primary porcine cell.
In some embodiments of any of the systems disclosed above, the modulator is an enzyme.
In some embodiments of any of the systems disclosed above, (i) the immortalized gene in the first polynucleotide sequence is flanked by a first pair of recombinase sites, and (ii) the replacement gene in the second polynucleotide sequence is flanked by a second pair of recombinase sites, and the modulator comprises a recombinase capable of causing an exchange.
In some embodiments, the first and second pairs of recombinase sites comprise Lox sites, and wherein the recombinase is Cre recombinase. In some embodiments, the first and second pairs of recombinase sites comprise FRT sites, and wherein the recombinase is a Flp recombinase. In some embodiments, the first and second pairs of recombinase sites comprise attP and attB sites, and wherein the recombinase is a PhiC31 recombinase.
In some embodiments, any of the systems disclosed herein further comprises a third polynucleotide sequence encoding a modulator, wherein the encoded modulator is capable of being activated upon induction of the system with an activator.
In some embodiments, the modulator is operably linked to an activator-inducible promoter of the third polynucleotide sequence. In some embodiments, the modulator is part of a fusion protein comprising an activator-binding domain. In some embodiments, the activator-binding domain is a ligand-binding domain of an estrogen receptor.
In some embodiments of any of the systems disclosed herein, the activator comprises tamoxifen (tamoxifen).
In some embodiments of any one of the systems disclosed herein, the immortalizing gene is (i) operably linked to a promoter, and (ii) flanked by the promoter and an in-frame termination sequence.
In some embodiments, the first polynucleotide sequence further comprises a reporter sequence, wherein the reporter sequence (i) is operably linked to a promoter, and (ii) flanks the promoter and an in-frame termination sequence.
In some embodiments, the immortalizing gene and the reporter sequence of the system are linked by a protease cleavage sequence.
In some embodiments of any one of the systems disclosed herein, the system further comprises a nucleic acid editing moiety comprising a nucleic acid cleavage moiety, wherein the nucleic acid editing moiety is capable of removing the immortalizing gene or fragment thereof from the first polynucleotide sequence to reduce the immortalizing gene in the cell.
In some embodiments of any one of the systems disclosed herein, the immortalizing gene is telomerase reverse transcriptase (TERT), SV 40T antigen, CDK4, HOXB, HOXA9, cMyc, Bmi1, or Myc T58A.
Another aspect of the disclosure provides a cell comprising any one of the systems described herein.
Another aspect of the disclosure provides a kit comprising one or more nucleic acid molecules encoding any of the systems described herein.
In one aspect, the present disclosure describes a method for modulating cell immortalization comprising: (a) expressing the system in a cell; and (b) inducing a system with the modulator to conditionally modulate the immortalization of the cell, wherein the system comprises: (i) a first polynucleotide sequence comprising an immortalizing gene, wherein expression of said immortalizing gene results in the immortalization of a cell; and (ii) a second polynucleotide sequence comprising a replacement gene replacing the immortalized gene or a fragment thereof, wherein expression of the immortalized gene in the cell is reduced upon induction of the system with a modulator that affects the exchange between the immortalized gene and the replacement gene.
In some embodiments, the first polynucleotide sequence is chromosomal.
In some embodiments, the method further comprises integrating the first polynucleotide sequence at one or more sites in the genome of the cell, wherein the one or more sites are selected from the group consisting of: AAVS1 site, GGTA1 site, CMAH site, B4GALNT2 site, B2M site, ROSA26 site, COLA1 site and TIGRE site.
In some embodiments, the method further comprises integrating the first polynucleotide sequence in the genome of the cell by a nucleic acid editing moiety comprising a nucleic acid cleavage moiety.
In some embodiments of any of the methods disclosed herein, the replacement gene is capable of enhancing allogeneic or xenogeneic compatibility of the cell.
In some embodiments, the replacement gene encodes a human copy selected from the group consisting of: B2M, HLAE, CD47, PDL1, CD46, CD55, CD59, A20, THBD, TFPI, CD39, EPCR, FASL, CIITA, CTLA-Ig and HO-1.
In some embodiments of any of the methods disclosed herein, the cell is incapable of expressing a functional copy of an incompatible allogeneic or xenogeneic epitope.
In some embodiments, the method further comprises modifying incompatible allogeneic or xenogeneic epitopes in the cell.
In some embodiments, the incompatible allogeneic or xenogeneic epitope is selected from: GGTA, CMAH, and B4 Gal.
In some embodiments of any of the methods disclosed herein, the cell is a non-human mammalian cell.
In some embodiments, the cell is a primary porcine cell.
In some embodiments of any of the methods disclosed herein, the modulator is an enzyme.
In some embodiments of any of the methods disclosed herein, (i) the immortalized gene in the first polynucleotide sequence is flanked by a first pair of recombinase sites, and (ii) the replacement gene in the second polynucleotide sequence is flanked by a second pair of recombinase sites, and the modulator comprises a recombinase capable of causing an exchange.
In some embodiments, the first and second pairs of recombinase sites comprise Lox sites, and wherein the recombinase is Cre recombinase.
In some embodiments, the first and second pairs of recombinase sites comprise FRT sites, and wherein the recombinase is a Flp recombinase.
In some embodiments, the first and second pairs of recombinase sites comprise attP and attB sites, and wherein the recombinase is a PhiC31 recombinase.
In some embodiments of any of the methods disclosed herein, the system further comprises a third polynucleotide sequence encoding a modulator, wherein the method further comprises inducing the cell with an activator to activate the encoded modulator.
In some embodiments, the modulator is operably linked to an activator-inducible promoter of the third polynucleotide sequence.
In some embodiments, the modulator is part of a fusion protein comprising an activator-binding domain.
In some embodiments, the activator-binding domain is a ligand-binding domain of an estrogen receptor.
In some embodiments of any of the methods disclosed herein, the activator comprises tamoxifen.
In some embodiments of any one of the methods disclosed herein, the immortalizing gene is (i) operably linked to a promoter, and (ii) flanked by the promoter and an in-frame termination sequence.
In some embodiments, the first polynucleotide sequence further comprises a reporter sequence, wherein the reporter sequence (i) is operably linked to a promoter, and (ii) flanks the promoter and an in-frame termination sequence.
In some embodiments, the immortalizing gene and the reporter sequence are linked by a protease cleavage sequence.
In some embodiments of any of the methods disclosed herein, the system further comprises a nucleic acid editing moiety comprising a nucleic acid cleavage moiety, wherein the nucleic acid editing moiety is capable of removing the immortalizing gene or fragment thereof from the first polynucleotide sequence to reduce the immortalizing gene in the cell.
In some embodiments of any of the methods disclosed herein, the immortalizing gene is telomerase reverse transcriptase (TERT) or Myc T58A.
In one aspect, the present disclosure describes a system for genetically modifying a cell, comprising: a polynucleotide sequence comprising an immortalizing gene, wherein expression of said immortalizing gene results in the immortalization of a cell, and wherein expression of said immortalizing gene is inactivated by an inactivating moiety; and a nucleic acid editing moiety comprising a nucleic acid cleavage moiety, wherein the nucleic acid editing moiety is capable of inducing at least one genetic modification in the cell, wherein the at least one genetic modification enhances allogeneic or xenogeneic compatibility of the cell, wherein induction of the inactivation moiety inactivates expression of the immortalizing gene in the cell after the at least one genetic modification in the cell.
In some embodiments, the at least one genetic modification is a germline modification.
In some embodiments, the nucleic acid editing moiety is configured to complex with a target polynucleotide in a cell to induce at least one genetic modification in the cell.
In some embodiments, the immortalizing gene in the polynucleotide sequence is flanked by a pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of deleting the immortalizing gene or fragment thereof from the polynucleotide sequence.
In some embodiments, the immortalizing gene in the polynucleotide sequence is in an active orientation and is flanked by a pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of inverting the immortalizing gene or fragment thereof into an inactivating orientation.
In some embodiments, (i) the immortalizing gene in the polynucleotide sequence is flanked by a pair of recombinase sites, and (ii) the system further comprises a further polynucleotide sequence comprising a replacement gene flanked by a further pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of causing an exchange between the immortalizing gene or fragment thereof and the replacement gene.
In some embodiments, the pair of recombinase sites and the other pair of recombinase sites comprise Lox sites, and wherein the recombinase is Cre recombinase.
In some embodiments, the pair of recombinase sites and the other pair of recombinase sites comprise FRT sites, and wherein the recombinase is a Flp recombinase.
In some embodiments, the pair of recombinase sites and the other pair of recombinase sites comprise attP and attB sites, and wherein the recombinase is a PhiC31 recombinase.
In some embodiments, (i) the polynucleotide sequence further comprises a pair of recombinase sites flanked by a promoter and an immortalizing gene, and (ii) the system further comprises a further polynucleotide sequence comprising a termination sequence flanked by a further pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of inserting the termination sequence between the pair of recombinase sites.
In some embodiments of any of the systems disclosed herein, the system further comprises a second polynucleotide sequence encoding a recombinase, wherein the encoded recombinase is capable of being activated upon induction of the system with an activator.
In some embodiments, the recombinase enzyme is operably linked to an activator-inducible promoter of the second polynucleotide sequence.
In some embodiments, the recombinase is part of a fusion protein comprising an activator-binding domain.
In some embodiments, the activator-binding domain is a ligand-binding domain of an estrogen receptor.
In some embodiments of any of the systems disclosed herein, the activator comprises tamoxifen.
In some embodiments of any one of the systems disclosed herein, the inactivating moiety comprises an additional nucleic acid editing moiety comprising an additional nucleic acid cleavage moiety, wherein the additional nucleic acid editing moiety is capable of removing the immortalizing gene or fragment thereof from the polynucleotide sequence to inactivate expression of the immortalizing gene in the cell.
In some embodiments of any one of the systems disclosed herein, the polynucleotide sequence is chromosomal.
In some embodiments, the polynucleotide sequence is integrated at one or more sites in the genome of the cell, wherein the one or more sites are selected from the group consisting of: AAVS1 site, GGTA1 site, CMAH site, B4GALNT2 site, B2M site, ROSA26 site, COLA1 site and TIGRE site.
In some embodiments, the polynucleotide sequence is integrated in the genome of the cell by a nucleic acid editing moiety comprising a nucleic acid cleavage moiety.
In some embodiments of any one of the systems disclosed herein, the at least one genetic modification comprises deletion of a gene or fragment thereof encoding an incompatible allogeneic or xenogeneic epitope in the cell.
In some embodiments, the incompatible allogeneic or xenogeneic epitope is selected from: GGTA, CMAH, and B4 Gal.
In some embodiments of any one of the systems disclosed herein, the at least one genetic modification comprises addition of a gene encoding a human copy selected from the group consisting of: B2M, HLAE, CD47, PDL1, CD46, CD55, CD59, A20, THBD, TFPI, CD39, EPCR, FASL, CIITA, CTLA-Ig and HO-1.
In some embodiments of any one of the systems disclosed herein, the cell is a non-human mammalian cell.
In some embodiments, the cell is a primary porcine cell.
In some embodiments of any one of the systems disclosed herein, the immortalizing gene is telomerase reverse transcriptase (TERT) or Myc T58A.
In some embodiments of any one of the systems disclosed herein, the at least one genetic modification comprises two or more different genetic modifications resulting from two or more genetic modification events.
In some embodiments, the two or more different genetic modifications comprise (i) knocking out a first gene encoding a first polypeptide, and (ii) knocking in a second gene encoding a second polypeptide.
In some embodiments, the second gene is a transgene.
In another aspect, the present disclosure provides a cell comprising any one of the systems described herein.
In another aspect, the present disclosure provides a kit comprising one or more nucleic acid molecules encoding any of the systems described herein.
In one aspect, the present disclosure describes a method of genetically modifying a cell, comprising: (a) expressing the system in a cell to induce at least one genetic modification in the cell; and (b) inducing an inactive moiety in the cell to conditionally modulate immortalization of the cell, wherein the system comprises: (i) a polynucleotide sequence comprising an immortalizing gene, wherein expression of the immortalizing gene results in immortalization of the cell, and wherein expression of the immortalizing gene is inactivated by an inactivating moiety, and (ii) a nucleic acid editing moiety comprising a nucleic acid cleavage moiety, wherein the nucleic acid editing moiety is capable of inducing at least one genetic modification in the cell, wherein the at least one genetic modification enhances allo-or xeno-compatibility of the cell, wherein induction of the inactivating moiety inactivates expression of the immortalizing gene in the cell after the at least one genetic modification in the cell.
In some embodiments, the at least one genetic modification is a germline modification.
In some embodiments, the nucleic acid editing moiety is configured to complex with a target polynucleotide in a cell to induce at least one genetic modification in the cell.
In some embodiments, the immortalizing gene in the polynucleotide sequence is flanked by a pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of deleting the immortalizing gene or fragment thereof from the polynucleotide sequence.
In some embodiments, the immortalizing gene in the polynucleotide sequence is in an active orientation and is flanked by a pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of inverting the immortalizing gene or fragment thereof into an inactivating orientation.
In some embodiments, (i) the immortalizing gene in the polynucleotide sequence is flanked by a pair of recombinase sites, and (ii) the system further comprises a further polynucleotide sequence comprising a replacement gene flanked by a further pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of causing an exchange between the immortalizing gene or fragment thereof and the replacement gene.
In some embodiments, the pair of recombinase sites and the other pair of recombinase sites comprise Lox sites, and wherein the recombinase is Cre recombinase.
In some embodiments, the pair of recombinase sites and the other pair of recombinase sites comprise FRT sites, and wherein the recombinase is a Flp recombinase.
In some embodiments, the pair of recombinase sites and the other pair of recombinase sites comprise attP and attB sites, and wherein the recombinase is a PhiC31 recombinase.
In some embodiments, (i) the polynucleotide sequence further comprises a pair of recombinase sites flanked by a promoter and an immortalizing gene, and (ii) the system further comprises a further polynucleotide sequence comprising a termination sequence flanked by a further pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of inserting the termination sequence between the pair of recombinase sites.
In some embodiments of any of the methods disclosed herein, the system further comprises a second polynucleotide sequence encoding a recombinase enzyme, wherein the encoded recombinase enzyme is capable of being activated upon induction of the system with an activator.
In some embodiments, the recombinase enzyme is operably linked to an activator-inducible promoter of the second polynucleotide sequence.
In some embodiments, the recombinase is part of a fusion protein comprising an activator-binding domain.
In some embodiments, the activator-binding domain is a ligand-binding domain of an estrogen receptor.
In some embodiments of any of the methods disclosed herein, the activator comprises tamoxifen.
In some embodiments of any one of the methods disclosed herein, the inactivating moiety comprises an additional nucleic acid editing moiety comprising an additional nucleic acid cleavage moiety, wherein the additional nucleic acid editing moiety is capable of removing the immortalizing gene or fragment thereof from the polynucleotide sequence to inactivate expression of the immortalizing gene in the cell.
In some embodiments of any of the methods disclosed herein, the polynucleotide sequence is chromosomal.
In some embodiments, the method further comprises integrating the polynucleotide sequence at one or more sites in the genome of the cell, wherein the one or more sites are selected from the group consisting of: AAVS1 site, GGTA1 site, CMAH site, B4GALNT2 site, B2M site, ROSA26 site, COLA1 site and TIGRE site.
In some embodiments, the method further comprises integrating the polynucleotide sequence in the genome of the cell by a nucleic acid editing moiety comprising a nucleic acid cleavage moiety.
In some embodiments of any of the methods disclosed herein, the at least one genetic modification comprises deletion of a gene or fragment thereof encoding an incompatible allogeneic or xenogeneic epitope in the cell.
In some embodiments, the incompatible allogeneic or xenogeneic epitope is selected from: GGTA, CMAH, and B4 Gal.
In some embodiments of any one of the methods disclosed herein, the at least one genetic modification comprises addition of a gene encoding a human copy selected from the group consisting of: B2M, HLAE, CD47, PDL1, CD46, CD55, CD59, A20, THBD, TFPI, CD39, EPCR, FASL, CIITA, CTLA-Ig and HO-1.
In some embodiments of any of the methods disclosed herein, the cell is a non-human mammalian cell.
In some embodiments, the cell is a primary porcine cell.
In some embodiments of any of the methods disclosed herein, the immortalizing gene is telomerase reverse transcriptase (TERT) or Myc T58A.
In some embodiments of any of the methods disclosed herein, the at least one genetic modification comprises two or more different genetic modifications resulting from two or more genetic modification events.
In some embodiments of any one of the methods disclosed herein, the two or more different genetic modifications comprise (i) knocking out a first gene encoding a first polypeptide, and (ii) knocking in a second gene encoding a second polypeptide.
In some embodiments, the second gene is a transgene.
In one aspect, the present disclosure describes a method of making a cell comprising two or more different genetic modifications resulting from two or more genetic modification events, the method comprising: (a) introducing into a cell a system comprising a polynucleotide sequence comprising an immortalizing gene, wherein expression of said immortalizing gene results in immortalization of the cell; and (b) performing two or more genetic modification events in the cell, wherein the two or more genetic modification events shorten the lifespan of the cell in the absence of the system.
In some embodiments, the two or more genetic modification events comprise two or more sequential rounds of genetic modification.
In some embodiments, the at least one genetic modification is a germline modification.
In some embodiments, the two or more different genetic modifications comprise (i) knocking out a first gene and (ii) knocking in a second gene.
In some embodiments, the first gene encodes an incompatible allogeneic or xenogeneic epitope of the cell or a fragment thereof.
In some embodiments, the incompatible allogeneic or xenogeneic epitope is selected from: GGTA, CMAH, and B4 Gal.
In some embodiments, the second gene is a transgene.
In some embodiments, the gene encodes a polypeptide capable of enhancing the allogeneic or xenogeneic compatibility of the cell.
In some embodiments, the polypeptide is a human copy selected from the group consisting of: B2M, HLAE, CD47, PDL1, CD46, CD55, CD59, A20, THBD, TFPI, CD39, EPCR, FASL, CIITA, CTLA-Ig and HO-1.
In some embodiments of any of the methods disclosed herein, the method comprises, in (b), inducing one or more effector moieties (activator moieties) into the cell, each of the one or more effector moieties comprising a nuclease, wherein the one or more effector moieties are capable of two or more genetic modification events.
In some embodiments of any one of the methods disclosed herein, the expression of the immortalizing gene is inactivated by an inactivating moiety, and the method further comprises, after (b), inducing the inactivating moiety into the cell to inactivate the immortalizing gene in the cell.
In some embodiments, the immortalizing gene in the polynucleotide sequence is flanked by a pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of deleting the immortalizing gene or fragment thereof from the polynucleotide sequence.
In some embodiments, the immortalizing gene in the polynucleotide sequence is in an active orientation and is flanked by a pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of inverting the immortalizing gene or fragment thereof into an inactivating orientation.
In some embodiments, (i) the immortalizing gene in the polynucleotide sequence is flanked by a pair of recombinase sites, and (ii) the system further comprises a further polynucleotide sequence comprising a replacement gene flanked by a further pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of causing an exchange between the immortalizing gene or fragment thereof and the replacement gene.
In some embodiments, the pair of recombinase sites and the other pair of recombinase sites comprise Lox sites, and wherein the recombinase is Cre recombinase.
In some embodiments, the pair of recombinase sites and the other pair of recombinase sites comprise FRT sites, and wherein the recombinase is a Flp recombinase.
In some embodiments, the pair of recombinase sites and the other pair of recombinase sites comprise attP and attB sites, and wherein the recombinase is a PhiC31 recombinase.
In some embodiments, (i) the polynucleotide sequence further comprises a pair of recombinase sites flanked by a promoter and an immortalizing gene, and (ii) the system further comprises a further polynucleotide sequence comprising a termination sequence flanked by a further pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of inserting the termination sequence between the pair of recombinase sites.
In some embodiments of any of the methods disclosed herein, the system further comprises a second polynucleotide sequence encoding a recombinase enzyme, wherein the encoded recombinase enzyme is capable of being activated upon induction of the system with an activator.
In some embodiments, the recombinase enzyme is operably linked to an activator-inducible promoter of the second polynucleotide sequence.
In some embodiments, the recombinase is part of a fusion protein comprising an activator-binding domain.
In some embodiments, the activator-binding domain is a ligand-binding domain of an estrogen receptor.
In some embodiments of any of the methods disclosed herein, the activator comprises tamoxifen.
In some embodiments of any one of the methods disclosed herein, the inactivating moiety comprises an additional nucleic acid editing moiety comprising an additional nucleic acid cleavage moiety, wherein the additional nucleic acid editing moiety is capable of removing the immortalizing gene or fragment thereof from the polynucleotide sequence to inactivate expression of the immortalizing gene in the cell.
In some embodiments of any of the methods disclosed herein, the cell is a non-human mammalian cell.
In some embodiments, the cell is a primary porcine cell.
In some embodiments of any of the methods disclosed herein, the immortalizing gene is telomerase reverse transcriptase (TERT) or Myc T58A.
In some embodiments of any one of the methods disclosed herein, the two or more different genetic modifications occur in the nucleus of a cell, and the method further comprises: genetically modified embryos are prepared by introducing the nucleus or a derivative thereof into an embryo or embryonic precursor.
In some embodiments, the embryonic precursor is an enucleated egg, wherein the introduction of a nucleus into the enucleated egg produces a genetically modified embryo.
In some embodiments, the embryo is a blastocyst, wherein the preparation of the genetically modified embryo is performed by introducing the cell or derivative thereof into the inner cell mass of the blastocyst.
In some embodiments of any of the methods disclosed herein, the method further comprises developing the genetically modified embryo into a transgenic animal comprising at least one genetic modification.
In some embodiments, the present disclosure provides genetically modified embryos made by any of the methods described herein.
In one aspect, the present disclosure describes a method of making a genetically modified embryo comprising: (a) expressing in a cell a system to induce at least one genetic modification in the nucleus of a cell, wherein the system comprises: (i) a polynucleotide sequence comprising an immortalizing gene, wherein expression of the immortalizing gene results in immortalization of the cell and wherein expression of the immortalizing gene is inactivated by an inactivating moiety, and (ii) a nucleic acid editing moiety comprising a nucleic acid cleavage moiety, wherein said nucleic acid editing moiety is capable of inducing at least one genetic modification in the nucleus of the cell, wherein introduction of the inactivating moiety inactivates expression of the immortalizing gene in the cell after the at least one genetic modification in the cell; (b) introducing an inactivating moiety to inactivate cell immortalization; and (c) preparing a genetically modified embryo by introducing the nucleus or derivative thereof into the embryo or embryonic precursor.
In some embodiments, the embryonic precursor is an enucleated egg, wherein the introduction of a nucleus into the enucleated egg produces a genetically modified embryo.
In some embodiments, the embryo is a blastocyst, wherein the preparation of the genetically modified embryo is performed by introducing the cell or derivative thereof into the inner cell mass of the blastocyst.
In some embodiments of any of the methods disclosed herein, the method further comprises developing the genetically modified embryo into a transgenic animal comprising at least one genetic modification.
In some embodiments of any of the methods disclosed herein, the at least one genetic modification is a germline modification.
In some embodiments of any of the methods disclosed herein, the inactivation moiety is a recombinase capable of inducing inactivation of the immortalizing gene by: (i) deletion of the immortalizing gene or fragment thereof, (ii) inversion of the immortalizing gene or fragment thereof, (iii) exchange of the immortalizing gene or fragment thereof with another polynucleotide sequence, or (iv) insertion of a termination sequence upstream of the immortalizing gene.
In some embodiments of any one of the methods disclosed herein, the inactivating moiety is a further nucleic acid editing moiety comprising a further nucleic acid cleavage moiety, wherein the further nucleic acid editing moiety is capable of removing the immortalizing gene or fragment thereof from the polynucleotide sequence.
In some embodiments of any of the methods disclosed herein, the polynucleotide sequence is chromosomal.
In some embodiments, the method further comprises integrating the polynucleotide sequence at one or more sites in the genome of the cell, wherein the one or more sites are selected from the group consisting of: AAVS1 site, GGTA1 site, CMAH site, B4GALNT2 site, B2M site, ROSA26 site, COLA1 site and TIGRE site.
In some embodiments, the method further comprises integrating the polynucleotide sequence in the genome of the cell by a nucleic acid editing moiety comprising a nucleic acid cleavage moiety.
In some embodiments of any of the methods disclosed herein, the at least one genetic modification comprises deletion of a gene or fragment thereof encoding an incompatible allogeneic or xenogeneic epitope in the cell.
In some embodiments, the incompatible allogeneic or xenogeneic epitope is selected from: GGTA, CMAH, and B4 Gal.
In some embodiments of any one of the methods disclosed herein, the at least one genetic modification comprises addition of a gene encoding a human copy selected from the group consisting of: B2M, HLAE, CD47, PDL1, CD46, CD55, CD59, A20, THBD, TFPI, CD39, EPCR, FASL, CIITA, CTLA-Ig and HO-1.
In some embodiments of any of the methods disclosed herein, the cell is a non-human mammalian cell.
In some embodiments, the cell is a primary porcine cell.
In some embodiments, the immortalizing gene is telomerase reverse transcriptase (TERT) or Myc T58A.
In another aspect, the present disclosure provides genetically modified embryos made by any of the methods disclosed herein.
Other aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the disclosure is capable of other and different embodiments and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.
Is incorporated by reference
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
Drawings
The novel features believed characteristic of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
FIG. 1 schematically illustrates an example of the vector system for reversible cell immortalization of the present invention. FIG. 1A schematically illustrates an exemplary polynucleotide construct comprising the immortalizing gene Tert flanked by loxP sites. The Tert gene is placed under the control of a promoter EF1 alpha; in frame with a termination sequence that is a 3' -poly a sequence; in-frame with the coding sequence of a reporter gene (EGFP), which is located 5' to the TERT sequence; and has a sequence encoding a cleavable peptide T2A between the two coding sequences. FIG. 1B schematically illustrates that the nucleic acid construct shown in FIG. 1A can be inserted into the genome at a target location, for example, between two endogenous genes (e.g., genes encoding GGTA).
Detailed Description
SUMMARY
A cell (e.g., a mammalian cell) can be genetically modified to introduce or inhibit a gene of interest in the cell. Some cases may require the simultaneous introduction and suppression (e.g., by deletion) of one or more genes or nucleic acid portions thereof, and may even require a combination of any of the above to obtain a cell with the desired properties. In order for a gene editing change to be stable and effective (e.g., a gene change may persist for at least the life of a cell), one or more editing operations may need to be performed at the genomic level.
In addition, obtaining genetically engineered cells with desired properties can involve multiple rounds of gene editing events on the cell. Multiple rounds of gene editing events on the same cell can lead to cell death and/or the expected null chromosomal changes in gene editing. Primary cells can undergo a limited number of cell divisions before entering the senescence phase. In some cases, multiple rounds of gene editing can shorten the lifespan of primary cells. In some cases, the lifespan of a primary cell may be too short to allow multiple rounds of gene editing before the cell enters the senescence stage.
One approach to solving this problem is to immortalize the cells. Immortalization can extend the life span of cells and enhance the proliferative capacity of cells. The immortalized cells can retain one or more of the physiological properties of the original cell while being able to undergo multiple rounds of cell division, typically indefinitely.
In some cases, immortalized cells behave similarly to transformed cells and thus can be tumorigenic when introduced into an organism.
In view of the above, there is a great need for alternative methods and systems involving gene editing, which involve multiple interventions in the genome of a cell. Thus, described herein are methods and systems for modulating cell immortalization. The cells can be useful in a variety of therapeutic applications, including, for example, the production of transgenic animals.
While various embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention herein. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.
The practice of some of the methods disclosed herein, unless otherwise indicated, employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4 th edition (2012); the series Current Protocols in Molecular Biology (edited by F.M. Ausubel et al); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor editor (1995)), Harlow and Lane editor (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic technology and Specialized Applications, 6 th edition (R.I. Freeney editor (2010)).
Definition of
The terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
In this application, the use of "or" means "and/or" unless stated otherwise. As used herein, the terms "and/or" and "any combination thereof" and grammatical equivalents thereof are used interchangeably. These terms may be expressed with any combination specifically contemplated. For illustrative purposes only, the following phrases "A, B and/or C" or "A, B, C, or any combination thereof" may mean "a alone; b alone; c alone; a and B; b and C; a and C; and A, B and C ". The term "or" may be used in combination or separately unless the context clearly indicates separate use.
The term "about" or "approximately" can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within 1 or greater than 1 standard deviation, depending on the practice of the given value. Alternatively, "about" may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or methods, the term can mean within an order of magnitude of the value, within 5 times the value, and more preferably within 2 times the value. Where a particular value is described in the application and claims, unless otherwise stated, the term "about" should be considered to mean an acceptable error range for that particular value.
As used in this specification and claims, the word "comprising" (and any form of comprising, such as "comprises and comprises"), "having" (and any form of having, such as "has and has"), "including" (and any form of including, such as "includes and includes") or "containing" (and any form of containing, such as "contains and includes") is inclusive or open-ended and does not exclude other unrecited elements or method steps. It is contemplated that any embodiment described in this specification can be practiced with respect to any method or composition of the present disclosure, and vice versa. In addition, the compositions of the present disclosure can be used to implement the methods of the present disclosure.
Reference in the specification to "some embodiments," "one embodiment," or "other embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the disclosure. To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.
As used herein, "cell" may refer to a biological cell. A cell may be the basic structural, functional and/or biological unit of a living organism. The cells may be derived from any organism having one or more cells. Some non-limiting examples include: prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotic organisms, protozoal cells, cells from plants (e.g., cells from plant crops, fruits, vegetables, cereals, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, lycopodium, horny bolts, liverworts, mosses), algal cells (e.g., cells of Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, gulfweed (Sargassum patens), c Echinoderm, nematode, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), and the like. Sometimes, the cells are not derived from a natural organism (e.g., the cells may be synthetically prepared, sometimes also referred to as artificial cells).
As used herein, the term "nucleotide" refers to a combination of bases-sugar-phosphates. The nucleotides may comprise synthetic nucleotides. The nucleotides may comprise synthetic nucleotide analogs. Nucleotides can be monomeric units of a nucleic acid sequence, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The term nucleotide may include ribonucleoside triphosphates-Adenosine Triphosphate (ATP), Uridine Triphosphate (UTP), Cytosine Triphosphate (CTP), Guanosine Triphosphate (GTP), and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives may include, for example, [ α S ] dATP, 7-deaza-dGTP and 7-deaza-dATP and nucleotide derivatives, which confer nuclease resistance to the nucleic acid molecules containing them. The term nucleotide as used herein may refer to dideoxyribonucleoside triphosphates (ddNTPs) and derivatives thereof. Illustrative examples of dideoxyribonucleoside triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. The nucleotides may be unlabeled or detectably labeled by well-known techniques. Labeling can also be performed with quantum dots. Detectable labels may include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels for nucleotides may include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7' -dimethoxy-4 '5-dichloro-6-carboxyfluorescein (JOE), rhodamine (rhodamine), 6-carboxyrhodamine (R6G), N, NcN' -tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-Rhodamine (ROX), 4- (4 'dimethylaminophenylazo) benzoic acid (DABCYL), waterfall Blue (Cascade Blue), Oregon Green (Oregon Green), Texas Red (Texas Red), Cyanine (Cyanine), and 5- (2' -aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS). Specific examples of fluorescently labeled nucleotides can include [ R6G ] dUTP, [ TAMRA ] dUTP, [ R110] dCTP, [ R6G ] dCTP, [ TAMRA ] dCTP, [ JOE ] ddATP, [ R6G ] ddATP, [ FAM ] ddCTP, [ R110] ddCTP, [ TAN1RA ] ddGTP, [ ROX ] ddTTP, [ dR6G ] ddATP, [ dR110] ddCTP, [ dTAMRA ] ddGTP and [ dROX ] ddTTP available from Perkin Elmer, Foster City, Calif.; FluoroLink deoxynucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, Ill.; fluorescein-15-dATP, fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, TR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2' -dATP available from Boehringer Mannheim, Indianapolis, Ind; and those available from Molecular Probes, Eugene, the chromosome-labeled nucleotide of Oreg., BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, waterfall blue-7-UTP, waterfall blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon green 488-5-dUTP, rhodamine green-5-UTP, rhodamine green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas-5-dUTP, and Texas-12-dUTP. Nucleotides may also be labeled by chemical modification. The chemically modified mononucleotide may be biotin-dNTP. Some non-limiting examples of biotinylated dNTPs may include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-cICTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-1.6-dUTP, biotin-20-dUTP).
The terms "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or analogs thereof, whether single-stranded, double-stranded or multi-stranded. The polynucleotide may be exogenous or endogenous to the cell. The polynucleotide may be present in a cell-free environment. The polynucleotide may be a gene or a fragment thereof. The polynucleotide may be DNA. The polynucleotide may be RNA. The polynucleotide may have any three-dimensional structure and may serve any function, known or unknown. The polynucleotide may comprise one or more analogs (e.g., altered backbone, sugar, or nucleobases). Modifications to the nucleotide structure, if present, may be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acids, xenogenic nucleic acids, morpholino, locked nucleic acids, diol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to a sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, stevioside, and tetanoside. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, locus(s) defined from linkage analysis, exons, introns, messenger RNA (mrna), transfer RNA (trna), ribosomal RNA (rrna), short interfering RNA (sirna), short hairpin RNA (shrna), micrornas (mirna), ribozymes, eDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfdna) and cell-free RNA (cfrna), nucleic acid probes, and primers. The sequence of nucleotides may be interrupted by non-nucleotide components.
As used herein, the terms "target polynucleotide" and "target nucleic acid" refer to a nucleic acid or polynucleotide targeted by a nucleic acid editing moiety of the present disclosure. For example, a "target nucleic acid" can be targeted by a nucleic acid integration moiety comprising a nucleic acid cleavage moiety described herein. The target nucleic acid can be DNA. The target nucleic acid can be RNA. A target nucleic acid can refer to a chromosomal sequence or an extrachromosomal sequence (e.g., episomal sequence, minicircle sequence, mitochondrial sequence, chloroplast sequence, etc.). The target nucleic acid can be a nucleic acid sequence that can be unrelated to any other sequence in the nucleic acid sample by a single nucleotide substitution. The target nucleic acid can be a nucleic acid sequence that is not related to any other sequence in the nucleic acid sample by 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions. In some embodiments, the substitution may not occur within 5, 10, 15, 20, 25, 30, or 35 nucleotides of the 5' end of the target nucleic acid. In some embodiments, the substitution may not occur within 5, 10, 15, 20, 25, 30, 35 nucleotides of the 3' end of the target nucleic acid. Generally, the term "target sequence" refers to a nucleic acid sequence on a single strand of a target nucleic acid. The target sequence may be a portion of a gene, regulatory sequences, genomic DNA, cell-free nucleic acids including cfDNA and/or cfRNA, cDNA, fusion genes, and RNA including mRNA, miRNA, rRNA, and the like.
As used herein, the term "gene" refers to a nucleic acid (e.g., DNA, such as genomic DNA and cDNA) and its corresponding nucleotide sequence involved in encoding an RNA transcript. As used herein with respect to genomic DNA, the term includes spacer, non-coding, and regulatory regions, and may include 5 'and 3' ends. In some uses, the term encompasses transcribed sequences, including 5 'and 3' untranslated regions (5'-UTR and 3' -UTR), exons, and introns. In some genes, the transcribed region comprises an "open reading frame" that encodes a polypeptide. In some uses of this term, a "gene" comprises only the coding sequence required to encode a polypeptide (e.g., an "open reading frame" or "coding region"). In some cases, the gene does not encode a polypeptide, such as ribosomal RNA genes (rRNA) and transfer RNA (trna) genes. In some cases, the term "gene" includes not only transcribed sequences, but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers, and promoters. A gene may refer to an "endogenous gene" or a native gene that is in its natural location in the genome of an organism. A gene may refer to an "exogenous gene" or a non-native gene. A non-native gene may refer to a gene that is not normally found in the host organism, but is introduced into the host organism by gene transfer. A non-native gene may also refer to a gene that is not in its natural location in the genome of an organism. A non-native gene may also refer to a naturally occurring nucleic acid or polypeptide sequence (e.g., a non-native sequence) that comprises a mutation, insertion, and/or deletion.
The term "transgene" refers to any nucleic acid molecule introduced into a cell. The resulting cells after receiving the transgene are referred to as transgenic cells. A transgene may include a gene that is partially or completely heterologous (i.e., foreign) to the transgenic organism or cell, or may represent a gene that is homologous to an endogenous gene of the organism or cell. In some cases, a transgene includes any polynucleotide, such as a gene encoding a polypeptide or protein, a polynucleotide that is transcribed into an inhibitory polynucleotide, or a polynucleotide that is not transcribed (e.g., lacks expression control elements, such as a promoter that drives transcription).
The term "transfection" refers to the introduction of nucleic acids into cells by non-viral or viral based methods. The nucleic acid molecule may be a gene sequence encoding a complete protein or a functional part thereof. See, e.g., Sambrook et al, 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88.
The term "expression" refers to the process or processes of transcription (e.g., into mRNA or other RNA transcript) of a polynucleotide from a DNA template; and/or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide or protein. The transcripts and the encoded polypeptides may be collectively referred to as "gene products". If the polynucleotide is derived from genomic DNA, expression may comprise splicing of the mRNA in a eukaryotic cell. With respect to expression, "up-regulation" refers to an increase in the level of expression of a polynucleotide (e.g., RNA, such as mRNA) and/or polypeptide sequence relative to the level of expression of the polynucleotide (e.g., RNA, such as mRNA) and/or polypeptide sequence in the wild-type state, while "down-regulation" refers to a decrease in the level of expression of a polynucleotide (e.g., RNA, such as mRNA) and/or polypeptide sequence in the wild-type state. Expression of the transfected gene may occur transiently or stably in the cell. During "transient expression", transfected genes are not transferred to daughter cells during cell division. The expression of the gene is lost over time, since its expression is limited to transfected cells only. In contrast, stable expression of a transfected gene can result when the gene is co-transfected with another gene that confers a selective advantage on the transfected cells. Such a selective advantage may be resistance to certain toxins presented to the cells. In cases where expression of a transfected gene is desired, the present application contemplates the use of codon-optimized sequences. Examples of codon-optimized sequences may be sequences optimized for expression in a eukaryote (e.g., a human) (i.e., optimized for expression in a human), or for use in another eukaryote, animal, or mammal. Codon optimization for host species other than humans, or for specific organs, is known. In some embodiments, a coding sequence encoding a protein can be codon optimized for expression in a particular cell (e.g., a eukaryotic cell). Eukaryotic cells can be those derived from a particular organism, such as a plant or mammal, including but not limited to a human or non-human eukaryote described herein or an animal or mammal, such as a mouse, rat, rabbit, dog, livestock, or non-human mammal or primate. Codon optimization refers to a method of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon (e.g., about or greater than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of the native sequence with a codon that is more or most frequently used in a gene of the host cell while maintaining the native amino acid sequence. Various species exhibit specific biases for certain codons for particular amino acids. Codon bias (difference in codon usage between organisms) is often correlated with the translation efficiency of messenger rna (mRNA), which is believed to depend inter alia on the nature of the codons being translated and the availability of specific transfer rna (trna) molecules. The predominance of the selected tRNA in the cell typically reflects the codons most frequently used in peptide synthesis. Thus, genes can be tailored based on codon optimization to optimally express genes in a given organism. Codon Usage tables are readily available, such as "Codon Usage Database" available from www.kazusa.orjp/Codon/, and these tables can be modified in a number of ways. Computer algorithms are also available for optimizing codons of specific sequences for expression in specific host cells, e.g., Gene Forge (Aptagen; Jacobus, Pa.).
The terms "expression cassette", "expression construct" or "expression vector" refer to a nucleic acid that includes a nucleotide sequence, such as a coding sequence and a template sequence, as well as sequences necessary for expression of the coding sequence. The expression cassette may be viral or non-viral. For example, an expression cassette includes a nucleic acid construct that, when introduced into a host cell, results in transcription and/or translation of an RNA or polypeptide, respectively. An untranslated or untranslated antisense construct or sense construct is expressly included in this definition. One skilled in the art will recognize that the inserted polynucleotide sequence need not be identical, but may be only substantially similar to the gene sequence from which it is derived.
As used herein, "plasmid" refers to a non-viral expression vector, such as a nucleic acid molecule encoding a gene and/or regulatory elements necessary for gene expression. As used herein, "viral vector" refers to a nucleic acid of viral origin that is capable of transporting another nucleic acid into a cell. When present in an appropriate environment, a viral vector is capable of directing the expression of one or more proteins encoded by one or more genes carried by the vector. Examples of viral vectors include, but are not limited to, retroviral vectors, adenoviral vectors, lentiviral vectors, and adeno-associated viral vectors.
As used herein, the term "promoter" refers to a polynucleotide sequence capable of driving transcription of a coding sequence in a cell. Thus, promoters used in the polynucleotide constructs of the present disclosure include cis-acting transcription control elements as well as regulatory sequences involved in regulating or modulating the transcription time and/or rate of a gene. For example, a promoter may be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcriptional terminator, an origin of replication, a chromosomal integration sequence, 5 'and 3' untranslated regions, or an intron sequence, all of which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to effect (turn on/off, regulate, etc.) gene transcription. A "constitutive promoter" is a promoter capable of initiating transcription in almost all tissue types, whereas a "tissue-specific promoter" initiates transcription only in one or several specific tissue types. An "inducible promoter" is a promoter that initiates transcription only under specific environmental, developmental, or pharmaceutical or chemical conditions. Exemplary inducible promoters may be doxycycline (doxycline) or tetracycline (tetracyline) inducible promoters. The tetracycline-regulated promoter may be tetracycline-inducible or tetracycline-repressible, referred to as the tet-on and tet-off systems. the tet-regulated system relies on two components, a tetracycline-controlled regulator (also known as a transactivator) (tTA or rtTA) and a tTA/rtTA-dependent promoter that controls downstream cDNA expression in a tetracycline-dependent manner. tTA is a fusion protein comprising the repressor of the Tn10 tetracycline resistance operon of Escherichia coli (Escherichia coli) and the carboxy-terminal portion of protein 16 of herpes simplex virus (VP 16). The tTA-dependent promoter consists of a minimal RNA polymerase II promoter fused to a tet operator (tetO) sequence (an array of seven homologous operator sequences). This fusion converts the tet repressor into a strong transcriptional activator in eukaryotic cells. In the absence of tetracycline or its derivatives (e.g., polycyclocycline), tTA binds to the tetO sequence, thereby allowing transcriptional activation of the tTA-dependent promoter. However, tTA is unable to interact with its target, nor is transcription occurring in the presence of doxycycline. The tet system using tTA is called tet-OFF because tetracycline or doxycycline down-regulates transcription. In contrast, in the tet-ON system, random mutagenesis has been used to isolate a mutant form of tTA (referred to as rtTA). In contrast to tTA, rtTA does not function in the absence of doxycycline, but requires the presence of a ligand for transactivation.
As used herein, the term "operably linked" refers to a functional relationship between two or more nucleic acid segments. Generally, it refers to the functional relationship of transcriptional regulatory sequences to transcriptional sequences.
The term "termination sequence" refers to a nucleic acid sequence that is recognized by the polymerase of the host cell and results in the termination of transcription. The termination sequence is a DNA sequence that provides for termination of mRNA transcription, or termination of mRNA transcription of an upstream open reading frame and ribosome translation, at the 3' end of a natural or synthetic gene. Prokaryotic termination sequences typically contain a GC-rich region with two-sided symmetry, followed by an AT-rich sequence. A common termination sequence is the T7 termination sequence. A variety of termination sequences are known in the art and can be used in the nucleic acid constructs of the invention, including TINT3, TL13, TL2, TR1, TR2 and T6S termination signals derived from bacteriophage lambda, as well as termination signals derived from bacterial genes (e.g., the trp gene of E.coli).
The term "polyadenylation sequence" (also known as "poly A+Site "or" poly A+Sequence ") refers to a DNA sequence that directs the termination and polyadenylation of a nascent RNA transcript. Efficient polyadenylation of recombinant transcripts is desirable due to the lack of poly-A+The transcripts of the tail are generally unstable and degrade rapidly. Poly-A for use in expression vectors+The signal may be "heterologous" or "endogenous". Endogenous poly A+A signal is a signal that is naturally found at the 3' end of the coding region of a given gene in the genome. Heterologous poly A+A signal is a signal that is isolated from one gene and placed 3' to another gene, such as the coding sequence of a protein. Frequently used heterologous poly-A+The signal is SV40 poly A+A signal. SV40 Poly A+The signal is contained in the 237bp BamHI/BclI restriction fragment and directs termination and polyadenylation; many vectors contain SV40 poly A+A signal. Another frequently used heterologous poly-A+The signal is derived from a Bovine Growth Hormone (BGH) gene; BGH poly A+Signals can also be obtained on a number of commercially available vectors. Poly-A from herpes simplex virus thymidine kinase (HSV tk) gene+The signal also serves as a poly-A on many commercial expression vectors+A signal.Polyadenylation signals facilitate the transfer of RNA from within the nucleus to the cytoplasm and increase the cellular half-life of such RNA. Polyadenylation signals are present at the 3' -end of the mRNA.
The term "exon" refers to a nucleic acid sequence found in genomic DNA that is predicted bioinformatically and/or experimentally confirmed to contribute a contiguous sequence to a mature mRNA transcript.
The term "intron" refers to a sequence present in genomic DNA that is bioinformatically predicted and/or experimentally confirmed to not encode part or all of an expressed protein and is transcribed into an RNA (e.g., pre-mRNA) molecule under endogenous conditions, but which is spliced out of the endogenous RNA (e.g., pre-mRNA), and the RNA is then translated into a protein.
The term "splice acceptor site" refers to a sequence present in genomic DNA that is bioinformatically predicted and/or experimentally confirmed as an acceptor site during pre-mRNA splicing, which may include both identified and unidentified natural and artificially derived or derivable splice acceptor sites.
An "Internal Ribosome Entry Site (IRES)" refers to a nucleotide sequence that allows translation to be initiated independently of the 5' -end/cap, thereby increasing the likelihood that 2 proteins will be expressed from a single messenger rna (mrna) molecule. IRES are typically located in the 5' UTR of positive-stranded RNA viruses with non-blocked genomes.
The term "2A peptide" refers to a class of viral oligopeptides of 18-22 Amino Acids (AA) in length that mediate "cleavage" of polypeptides during translation in eukaryotic cells. The name "2A" refers to a specific region of the viral genome, and different viruses 2A are often named after the virus from which they are derived. The earliest 2A discovered was F2A (foot-and-mouth disease virus), after which E2A (equine rhinitis virus a virus), P2A (porcine teschovirus-12A) and T2A (bovine asigna virus 2A) were also identified. It is believed that the 2A-mediated "self-cleavage" mechanism is the ribosome skipping the formation of the glycyl-prolyl peptide bond at the C-terminus of the 2A sequence. The 2A peptide (like) sequence mediates the self-processing of the primary translation product through a variety of processes called "ribosome skipping", "stop-and-go" translation and "stop-and-go" translation. The 2A peptide (like) sequences are present in different groups of positive-and double-stranded RNA viruses, including the small rnaviridae (Picornaviridae), Flaviviridae (Flaviviridae), Tetraviridae (Tetraviridae), Dicistroviridae (dicystroviridae), Reoviridae (Reoviridae), and holistic viridae (Totiviridae).
The terms "complementary," "complement," "complementary," and "complementarity," as used herein, refer to sequences that are fully complementary to, and can hybridize to, a given sequence. In some cases, a sequence that hybridizes to a given nucleic acid is referred to as the "complement" or "reverse complement" of a given molecule if its base sequence on a given region is capable of complementarily binding to the base sequence of its binding partner, e.g., such that a-T, A-U, G-C and G-U base pairs are formed. In general, a first sequence that is hybridizable to a second sequence can specifically or selectively hybridize to the second sequence, such that hybridization to the second sequence or set of second sequences is preferred over hybridization to non-target sequences during the hybridization reaction (e.g., thermodynamically more stable under a given set of conditions, such as stringency conditions). Typically, hybridizable sequences share a degree of sequence complementarity over all or part of their respective lengths, such as 25% to 100% complementarity, including at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence complementarity. For example, to assess percent complementarity, sequence identity may be measured by any suitable alignment algorithm, including, but not limited to, the Needleman-Wunsch algorithm (see, e.g., embos Needle aligner (aligner) available at www.ebi.ac.uk/Tools/psa/embos _ Needle/nuclear. html), the BLAST algorithm (see, e.g., BLAST alignment tool available at BLAST, ncbi.nlm.nih.gov/blast.cgi, optionally with default settings), or the Smith-Waterman algorithm (see, e.g., embos Water aligner available at www.ebi.ac.uk/Tools/psa/embos _ Water/nuclear. html, optionally with default settings). Any suitable parameters of the selected algorithm, including default parameters, may be used to evaluate the optimal alignment.
Complementarity may be perfect or sufficient/sufficient. Perfect complementarity between two nucleic acids can mean that the two nucleic acids can form a duplex in which each base in the duplex binds to a complementary base through Watson-Crick pairing. Sufficient or sufficient complementarity may mean that the sequence in one strand is not completely and/or perfectly complementary to the sequence in the opposite strand, but that sufficient bonding occurs between the bases on both strands to form a stable hybridization complex under a set of hybridization conditions (e.g., salt concentration and temperature). The conditions can be used to predict the melting temperature (T) of the hybridized strands by using the sequences and standard mathematical calculationsm) Or by using conventional methods for TmEmpirical measurements were made to predict.
As used herein, the term "knock-out" (KO) refers to the deletion, inactivation, or excision of a gene in a cell or organism (e.g., a pig or other animal) or in any cell of a pig or other animal. As used herein, KO may also refer to a method in which a deletion, inactivation, or excision of a gene or portion thereof is or has been performed such that the protein encoded by the gene is no longer formed.
As used herein, the term "knock-in (KI)" refers to the addition, substitution, or mutation of a nucleotide of a gene in a pig or other animal or in any cell of a pig or other animal. As used herein, KI may also refer to a method of performing or having performed an addition, substitution, or mutation of nucleotides of a gene or portion thereof.
The terms "peptide", "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of at least two amino acid residues joined by peptide bonds. The term does not denote a particular length of polymer, nor is it intended to imply or distinguish whether the peptide is produced synthetically, chemically or enzymatically using recombinant techniques, or naturally occurring. The term applies to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acids. The term includes amino acid chains of any length, including full-length proteins, as well as proteins with or without secondary and/or tertiary structures (e.g., domains). The term also encompasses amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation (e.g., binding to a labeling component). The term "amino acid" as used herein refers to natural and unnatural amino acids, including but not limited to modified amino acids and amino acid analogs. Modified amino acids can include natural amino acids and unnatural amino acids that have been chemically modified to include groups or chemical moieties that do not naturally occur on the amino acid. Amino acid analogs can refer to amino acid derivatives. The term "amino acid" includes D-amino acids and L-amino acids.
The terms "derivative," "variant," and "fragment," when used herein with respect to a polypeptide, refer to the polypeptide as it relates to the wild-type polypeptide, e.g., in terms of amino acid sequence, structure (e.g., secondary and/or tertiary), activity (e.g., enzymatic activity), and/or function. Derivatives, variants, and fragments of the polypeptides may comprise one or more amino acid variants (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof, as compared to the wild-type polypeptide.
As used herein, the term "percent (%) identity" refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues of a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences can be omitted for comparison purposes). Alignments intended to determine percent identity can be performed in various ways within the skill of the art, for example, using publicly available computer software, such as BLAST, ALIGN, or megalign (dnastar) software. Percent identity between two sequences can be calculated by aligning the test sequence with the comparison sequence using BLAST, determining the number of amino acids or nucleotides in the aligned test sequence that are identical to the amino acids or nucleotides at the same positions in the comparison sequence, and dividing the number of identical amino acids or nucleotides by the number of amino acids or nucleotides in the comparison sequence.
As used herein, the term "nucleic acid editing moiety" refers to a moiety that can induce one or more gene edits in a polynucleotide sequence. The polynucleotide sequence may be in a host cell. Alternatively, the polynucleotide sequence may not be in the host cell (e.g., a reaction mixture). Nucleic acid editing using a nucleic acid editing portion can include introducing one or more heterologous nucleic acids (e.g., a gene or fragment thereof) in a cell, or deleting one or more endogenous nucleic acids (e.g., a gene or fragment thereof) from a cell. In some cases, gene editing using a nucleic acid editing portion can include replacing any one or more polynucleic acids (e.g., genes or fragments thereof) thereof with one or more replacement nucleic acids. In some cases, nucleic acid editing using a nucleic acid editing portion can comprise a combination of any of the above, either simultaneously or sequentially. In some cases, one or more polynucleic acids may be DNA (e.g., DNA polynucleotide sequences). In some cases, one or more polynucleic acids may be genomic DNA. In some cases, one or more genes, or nucleic acid portions thereof, can be added to or deleted from the chromosomal DNA of the cell by a nucleic acid editing moiety. In some cases, one or more polynucleic acids may be added to or deleted from the chromosomal DNA of a cell by a nucleic acid editing moiety. The one or more polynucleic acids may be heterologous to the cell. In some cases, one or more polynucleic acids may be contained in the exosomes. In some cases, one or more polynucleic acids may be in the mitochondria or any other organelle. In some cases, any one or more genes or nucleic acid portions thereof can be added to or deleted from the episomal or chromosomal DNA of the cell by a nucleic acid editing moiety. In some cases, one or more polynucleic acids may be RNA. In some cases, one or more exogenous polynucleic acids may be added to the genomic DNA by integrating the exogenous polynucleic acid into the genomic DNA. Any suitable method of having at least a nucleic acid editing portion can be used for integration of any one or more genes into the genome of a cell.
Non-limiting examples of nucleic acid editing moieties can include, but are not limited to, CRISPR-mediated gene-modifying polypeptides, such as Cas9, Cas12a (Cpf1), or other CRISPR endonucleases, Argonaute endonucleases, transcription activator-like (TAL) effectors and nucleases (TALENs), Zinc Finger Nucleases (ZFNs), expression vectors, transposon systems (e.g., PiggyBac transposases), or any combination thereof. Designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases can be used to generate target genomic perturbations.
Target genome editing can be through CRISPR-mediated genetic modification using a Cas endonuclease or a Cas-like endonuclease. CRISPRs (clustered regularly interspaced short palindromic repeats), also known as spiders (interlude direct repeats), constitute a family of DNA loci that are generally specific for a particular bacterial species. CRISPR loci contain a unique class of interspersed Short Sequence Repeats (SSRs) and related genes that are recognized in e. Similar dispersions can be identified in SSR in Haloferax mediterranei (Haloferax mediterranei), Streptococcus pyogenes (Streptococcus pyenes), Anabaena (Anabaena) and Mycobacterium tuberculosis (Mycobacterium tuberculosis). CRISPR loci differ from other SSRs generally by the structure of repeated sequences, known as short regularly interspaced repeats (SRSRs). The repetitive sequences are short elements in clusters regularly spaced by unique insertion sequences of substantially constant length. Although the repeat sequences are highly conserved among strains, the number of interspersed repeat sequences and spacer sequences typically varies from strain to strain. The CRISPR locus has been identified in more than 40 prokaryotic organisms including, but not limited to, pyrenophora ((Aeropyrum), pyrobacium (pyrobacterium), Sulfolobus (Sulfolobus), archaebacteria (archaoglobus), haloboxella (halocarpula), Methanobacterium (Methanobacterium), Methanococcus (Methanococcus), Methanosarcina (Methanosarcina), Pyrococcus (Methanopyrus), Pyrococcus (Pyrococcus), Corynebacterium (Corynebacterium), Mycobacterium (Mycobacterium), Streptomyces (Streptomyces), liquid producing bacteria (axx), Porphyromonas (Porphyromonas), chlorobacterium (chlorella), thermobacter (thermobacter), thermobacter (thermobacillus), thermophilic Bacillus (thermobacillus), Staphylococcus (Clostridium), Clostridium (Clostridium), rhodobacter (Clostridium), Clostridium (Clostridium) and Clostridium (Clostridium) bacteria) are used as a strain (Clostridium) bacteria) and Bacillus) in a. bacteria, pseudomonas) bacteria, pseudomonas (e, pseudomonas) bacteria, pseudomonas (e, pseudomonas) bacteria, pseudomonas) and Bacillus) bacteria, pseudomonas (e, pseudomonas) are included in a, pseudomonas (e, pseudomonas (Bacillus, pseudomonas) bacteria, pseudomonas) and Bacillus, pseudomonas (e, pseudomonas) in a, pseudomonas (e, pseudomonas) of a, pseudomonas (e, pseudomonas) of a, pseudomonas (Bacillus) of a, pseudomonas) of a, pseudomonas (e, pseudomonas, chromobacterium (Chromobacterium), Neisseria (Neisseria), Nitrosomonas (nitromonas), desulphatovibrio (Desulfovibrio), Geobacter (Geobacter), mucococcus (Myxococcus), Campylobacter (Campylobacter), wolflorus (wolinaella), Acinetobacter (Acinetobacter), Erwinia (Erwinia), Escherichia (Escherichia), Legionella (Legionella), Methylococcus (Methylococcus), Pasteurella (Pasteurella), Photobacterium (Photobacterium), Salmonella (Salmonella), Xanthomonas (yeranthomonas), Yersinia (Yersinia), Treponema (Treponema) and thermoplasma (thermoga).
The Cas9 gene can be found in several different bacterial genomes, typically in the same loci as Cas1, Cas2 and Cas4 genes, as well as in CRISPR cassettes. In addition, Cas9 protein contains a readily identifiable C-terminal region that is homologous to transposon ORF-B and contains an active RuvC-like nuclease, arginine-rich region. The Cas9 protein may be from an organism of the genus: streptococcus (Streptococcus), Campylobacter (Campylobacter), Nitiduracor, Staphylococcus (Staphylococcus), Microbacterium (Parvibacterium), Roseburia (Roseburia), Neisseria (Neisseria), Acetobacter gluconicum (Gluconobacter), Azospirillum (Azospirillum), Sphaerhagia, Lactobacillus (Lactobacilli), Eubacterium (Eubacterium) or Corynebacterium (Corynebacterium), Carnobacterium (Carnobacterium), Rhodobacterium (Rhodobacter), Listeria (Listeria), Paluobacter, Clostridium (Clostridium), Lachnospiraceae (Lachnospiraceae), Clostridium (Clostridium), Clostridium (Leptotrium), Franciella (Francisella), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (Clostridium), Clostridium (, Bacillus (Bacillus), Brevibacterium (Brevibacterium), Methylobacterium (Methylobacterium), or Aminococcus (Acidaminococcus).
The nucleic acid editing moiety may comprise a nucleic acid cleavage moiety. The nucleic acid cleavage moiety can introduce a break or cleavage in the nucleic acid site molecule. The nucleic acid cleavage moiety can, for example, be capable of recognizing a particular cleavage recognition site when located in proximity to the cleavage recognition site on the target polynucleotide sequence. In some cases, the nucleic acid cleavage portion can be guided by a second molecule (e.g., a nucleic acid, such as a sequence-specific guide RNA of Cas9) to a specific cleavage site on the polynucleic acid to introduce a break or cleavage on the polynucleic acid. The nucleic acid cleavage moiety may initiate the introduction, deletion or substitution of a nucleic acid in the genomic DNA. In some cases, the nucleic acid cleavage moiety is a nuclease or a functional fragment thereof. In some cases, the nucleic acid cleavage moiety can comprise an endonuclease, an exonuclease, a DNase, an RNase, a strand-specific nuclease or a more specialized nuclease (e.g., CRISPR-associated protein 9, Cas9), or any fragment thereof. In some cases, the nucleic acid cleaving moiety can be a nicking enzyme.
In some cases, the nuclease can be an AAV Rep protein, Rep 68/78. The AAV genome comprises an Inverted Terminal Repeat (ITR) that further comprises a Rep Binding Site (RBS) to which Rep68/78 can bind and introduce a gap. The integration site of AAV in the human genome, AAVs1, also comprises a 500bp region containing the site. Rep68/78 binds to cell fragments and acts as a bridge connecting AAV ITRs with AAVs 1. AAVS1 is critical for site-specific integration of genetic material by AAV and may be useful in the methods described herein.
The cleavage recognition site can be at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more contiguous nucleic acid sequences. The cleavage recognition site may be up to 10, 9, 8, 7, 6, 5, 4, 3 or 2 consecutive nucleic acid sequences. Alternatively, the cleavage recognition site may be a single nucleic acid (e.g., thymine (T) or adenine (a) or cytosine (C) or guanine (G)). The cleavage recognition site may be defined by the particular structure (e.g., folding) of the target polynucleotide sequence, and may not be dependent on any particular contiguous nucleic acid sequence within the target polynucleotide sequence. Alternatively, the cleavage recognition site may be defined by both the contiguous nucleic acid sequence and the specific structure of the target polynucleotide sequence.
The nucleic acid editing portion may comprise a viral mechanism (viral machinery) or fragment thereof capable of introducing viral genes into a host cell. For example, a nucleic acid editing moiety can refer to a viral integrase system, such as a lentiviral integrase system. Integrase is a retroviral enzyme that catalyzes the integration of DNA into the genome of mammalian cells, a useful step in the replication of a retrovirus during retroviral infection. The integration process can be divided into two sequential reactions. The first reaction, termed 3' -processing, corresponds to a specific endonuclease reaction that produces viral DNA ends that are competent for subsequent covalent insertion (termed strand transfer) into the host cell genome by transesterification reactions. In some cases, a nucleic acid editing portion may also refer to a transposon/transposase or a transposase system or components thereof for integrating a piece of DNA into a genome. However, insertion of foreign DNA into a particular genomic sequence is preferred over random and semi-random integration throughout the target cell genome (e.g., using certain retroviral vectors and transposon/transposases). Random and semi-random integration procedures can lead to outcomes such as position effect variation, transgene silencing, and in some cases, insertional mutagenesis due to transcriptional dysregulation or physical disruption of endogenous target cellular genes.
In some cases, the nucleic acid editing moiety can be a fusion polypeptide. The fusion polypeptide can comprise at least two members including (i) a first polypeptide that confers nuclease activity or a modification thereof (e.g., reduced or inactivated nuclease activity), and (ii) a second polypeptide that confers one or more additional activities selected from the group consisting of: methyltransferase activity, demethylase activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, polyadenylation activity, SUMO activity, dessumo activity, ribosylation activity, enucleation glycosylation activity, myristoylation activity, remodeling activity, protease activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, synthase activity, synthetase activity or demannylation activity. At least two members of the fusion polypeptide can be linked by a cleavage sequence (e.g., a self-cleaving peptide, such as P2A). Alternatively, at least two members of the fusion polypeptide may not be joined by any cleavage sequence.
The terms "recombinant" or "DNA recombination," as used interchangeably herein, generally refer to a method of exchanging nucleic acid fragments from two different polynucleotide sequences. Recombination may involve the breaking and exchange of DNA sequences between the two strands of DNA. Recombination can be regulated by recombinant moieties (e.g., small molecules or polypeptides, such as enzymes). In one example, recombination can be regulated by at least 1, 2, 3, 4, 5, or more enzymes. Recombination can be regulated by up to 5, 4, 3, 2, or 1 enzyme. The one or more enzymes that perform or promote DNA recombination may be a single or multiple enzymes that perform the following steps: creating a break (excision), bringing the exchange strand near the site of the break, and removing the preexisting strand, and sealing the broken end or connection. The one or more enzymes may comprise, for example, one or more endonucleases for generating a break; and one or more ligases for ligation. DNA recombination can be carried out by a recombinase. As used interchangeably herein, the terms "sequence-specific recombination" and "site-specific recombination" refer to functions performed by a recombination moiety (e.g., an enzyme) that is, for example, a recombinase that recognizes and binds to short nucleic acid sites or "sequence-specific recombinase target sites" (i.e., recombinase recognition sites) and catalyzes the recombination of nucleic acids associated with these sites. These enzymes may include recombinases, transposases, and integrases. The terms "sequence-specific recombinase target site", "site-specific recombinase target site", "sequence-specific target site", and "site-specific target site" refer to short nucleic acid sites or sequences, i.e., recombinase recognition sites, that are recognized by a sequence or site-specific recombinase and become exchange regions in a site-specific recombination event. Examples of sequence specific recombinase target sites include, but are not limited to, lox sites, att sites, dif sites, and frt sites. The Cre/lox system is often used for sequence-specific recombination of DNA. The Cre recombinase is a modulator of the Cre/lox system, which catalyzes site-specific recombination by exchange between two remotely spaced Cre recognition sequences (i.e., loxP sites). The loxP site refers to a nucleotide sequence of Cre recombinase, a product of Cre gene of bacteriophage P1, which catalyzes a site-specific recombination event. The loxP site comprises two 13bp inverted repeats separated by an 8bp spacer. Due to Cre-mediated recombination, any DNA sequence introduced between the two 34-bp loxP sites (called "floxed" DNA flanked by loxP sites) was excised. The presence of Cre recombinase is necessary for the exchange of the first and second polynucleotide sequences.
The term "replicative senescence" of a cell refers to the aging of a cell and the loss of cell replicative capacity. Normal mammalian diploid or primary cells arrest terminal growth due to replicative senescence, which may be a protective mechanism for an organism as it acts as a tumor suppressor mechanism. Replicative senescence is characterized by marked changes in cell morphology, gene expression and metabolism. Activation of tumor suppressor proteins such as p53, RB, and p16 are common gene expression changes. Metabolic changes are often associated with increased lysosomal biogenesis, as indicated by overexpression of endogenous β -galactosidase. The reduction in telomere length also marks the natural progression of the cell cycle to aging or senescence. Each chromosome in a cell has telomeres, which are repetitive DNA sequences at the ends of human chromosomes (e.g., TTAGGG). Telomeres function by preventing chromosomes from losing base pair sequences at their ends. They also prevent chromosomes from fusing to each other. Telomeres can be up to 15,000 base pairs in length. However, some telomeres are lost with each cell division (usually 25-200 base pairs are lost with each division). When telomeres become too short, chromosomes reach a "critical length" and cannot replicate any more. This means that the cells become "old" and die by a process called apoptosis. Telomere activity is controlled by two mechanisms: erosion and addition. Erosion occurs with each cell division, and addition depends on telomerase activity. During natural aging, telomerase activity is low or inhibited. Cell morphological changes are often associated with increased cell size and multinuclear development.
As used herein, the term "immortalization" refers to the process of producing one or more immortalized cells. The cells may be immortalized in vitro or in vivo. Immortalized cells are characterized as exhibiting enhanced proliferative capacity (e.g., unlimited proliferative capacity) and enhanced lifespan (e.g., indefinite lifespan of the cell) as compared to the absence of such immortalization. In some cases, cell immortalization may involve artificially turning over the natural terminal growth arrest and replicative senescence of cells and subjecting the cells to repeated cycles of cell division under, for example, in vitro cell culture conditions. In addition to enhanced proliferation and/or longevity characteristics, an immortalized cell may exhibit nearly identical characteristics (e.g., cell spreading, cell volume, cell tension, migration, expression of one or more genes, sternness or lack thereof, etc.) in each cell cycle as compared to the characteristics that it had prior to cell immortalization.
In some cases, the immortalized cells can exhibit an increase in the number of cell divisions of at least 0.1-fold, at least 0.2-fold, at least 0.3-fold, at least 0.4-fold, at least 0.5-fold, at least 1-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1000-fold, or more, as compared to similar cells that have not been immortalized (e.g., from the same lineage). In some embodiments, the immortalized cell is characterized by an increase in the number of cell cycles of at least 0.1-fold, at least 0.2-fold, at least 0.3-fold, at least 0.4-fold, at least 0.5-fold, at least 1-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1000-fold, or more, as compared to a similar cell that is not immortalized (e.g., from the same lineage). In some embodiments, the immortalized cells can be maintained in vitro (i.e., in culture) for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years, at least 3 years, or longer as compared to similar cells that have not been immortalized (e.g., from the same lineage).
In some cases, reliable immortalization may allow the formation of stable cell lines, i.e., cell characteristics that remain constant between each cycle, without undergoing differentiation, transdifferentiation, or otherwise changing any physiological state or characteristic over time.
Cell immortalization can involve inducing one or more immortalizing genes (e.g., activating one or more endogenous genes, introducing one or more heterologous genes, etc.) that, when expressed (e.g., transcribed into an RNA molecule and/or translated into a polypeptide molecule), are configured to effect immortalization of the host cell.
Examples of immortalizing genes may include, but are not limited to, polyoma virus T antigen, adenovirus E1A and E1B gene products, papillomavirus E6 and E7 proteins, hepatotrophic virus X protein, Epstein-Barr virus EBNA, LMP, BALF.1. protein, BARF1 protein, BCRF1 protein and BART gene product, herpesvirus K1 protein associated with Kaposi's sarcoma virus, K2 protein, K15 protein, ORF72 product, ORF74 product, LANA and carboposi (Kaposi), herpesvirus saimiri STP, TIP, ORF13, ORF72, ORF73 and ORF74 product, human T cell leukemia virus type 1 Tax and HBZ protein, hepatitis C virus core protein, NS3 and NS5A protein, Cip/Kip forms, Ink4, dominant negative members of the dominant cell leukemia virus type 1 Tax and HBZ protein, proteins such as the dominant cell cycle protein, Bolb protein, Bbldm protein, Bblk/Bblk protein, Bddn protein, CDK 23 protein and Bddn protein, such as the anti-necrosis factor-apoptosis protein, CDK, and Bolc protein, and Bddn protein, such as the like, cIAP1, cIAP2, ILP2, livin/MLIAP, NIAP, survivin, Al, Bcl-2, Bcl2-L-10, Bcl-B, Bcl-W, BC1-XL, Bfl-1, BOO/DIVA, Mcl-1, NR-13, vBcl-2, vIAP, C-FLIP, V-FLIP, adenovirus E3-6.7k protein, RIDa, RID6, E3-14.7k protein, African swine fever virus A179L [5-HL ] protein, baculovirus p35, cytomegalovirus 62.7, pUL37 [ vmIA ], UL36[ vICA ], vIRA, Epstein-Barr virus BHRF1 protein, fowl pox virus FPV039 protein, gamma MAP 68M8[ vMAP ] protein, propane virus type P4642, herpes virus, herpes simplex virus 4642, herpes virus, murine influenza virus HBsAPP 465, BCH 5-3, BCH 5-DNA 465, BCH 5-DNA 465 protein, BCH 5-DNA protein, BCH 5 protein, BCH-DNA-protein, DNA-protein, DNA-protein, DNA-protein, DNA-protein, DNA-protein, DNA, Influenza M2 herpesviruses associated with Kaposi's sarcoma, K7 protein, K13 protein, KSBcl-2, myxoma M11L, parapoxvirus ORFV125, vaccinia virus CrmaA/SPI-2, F1L, NIL, SPI-1), retroviral oncoproteins (e.g., V-Abl, V-Akt, V-Crk, V-Cyclin, V-ErbA, V-Erbb, V-Fos, V-Ha-ras, V-Jun, V-Ki-ras, V-Mos, V-Myc, V-P3K, V-Sis, and V-Src), telomerase reverse transcriptase (TERT), mutants of cellular oncogenes, tumor suppressor genes, functional variants thereof, modifications thereof, and combinations thereof. In some cases, one or more immortalizing genes can work in conjunction with a helper gene to cause cell immortalization.
The term "episomal" or "extrachromosomal" genomic component or nucleic acid refers to an extrachromosomal nucleic acid. An episomal nucleic acid can be an exogenous DNA that is physically independent of the endogenous chromosome of the cell. The episomal DNA can be plasmid DNA or nucleic acid derived from a viral vector. Examples include episomal linkers derived from, for example, adeno-associated virus (AAV) vectors. The episomal nucleic acid can be DNA. Free DNA may have replicative properties and may be transcribed into messenger RNA, which may then be further translated into protein. Other examples of episomes include, but are not limited to, insertion sequences and transposons, viral vectors, bacterial F-elements. Other types of exemplary extrachromosomal DNA can be organelle DNA, such as mitochondrial DNA, which encodes the thirteen genes required for energy metabolism. Another exemplary extrachromosomal DNA may be an extrachromosomal circular DNA that is a DNA repeat sequence derived from coding and non-coding regions of a chromosome.
The term "allogeneic" or "xenogeneic" refers to biological components, such as cells, nucleic acids, etc., from the organism's own or non-own donors, respectively. The term allogeneic or xenogeneic epitope refers to a T cell epitope that can activate a cytotoxic response against a cell having the epitope in a host organism into which the cell is implanted. When binding of an allogeneic or xenogeneic epitope to an MHC peptide is displayed on the cell surface, the epitope is recognized by a T Cell Receptor (TCR) expressed on the surface of a host T cell. The binding of the TCR to the chromosome of an epitope of an antigen that binds to the MHC activates host T cells and triggers a series of cytotoxic T cell responses, thereby destroying the cells bearing the epitope.
As used herein, "treatment" refers to any of the following: alleviating one or more symptoms of a disease (e.g., cancer); preventing such symptoms from occurring before they occur; slowing or completely preventing the progression of the disease (e.g., as evidenced by a longer interval between recurrent episodes, slowing or preventing worsening of symptoms, etc.); accelerating the onset of remission; slowing the irreversible damage caused in the progression of the disease-the chronic stage (in the primary and secondary stages); delaying the start of the progression phase; or any combination thereof.
The Internal Ribosome Entry Site (IRES) is a nucleotide sequence that allows translation to be initiated independently of the 5' -end/cap, thereby increasing the likelihood that 2 proteins will be expressed from a single messenger rna (mrna) molecule. IRES are typically located in the 5' UTR of positive-stranded RNA viruses with non-blocked genomes. Another method to express 2 proteins from a single mRNA molecule is to insert a 2A peptide (like) sequence between their coding sequences. The 2A peptide (like) sequence mediates the self-processing of the primary translation product by a variety of methods known as "ribosome skipping", "stop-and-go" translation and "stop-and-go" translation. The 2A peptide (like) sequences are present in different groups of positive-and double-stranded RNA viruses, including the small rnaviridae (Picornaviridae), Flaviviridae (Flaviviridae), Tetraviridae (Tetraviridae), Dicistroviridae (dicystroviridae), Reoviridae (Reoviridae), and holistic viridae (Totiviridae).
As used herein, "administration" and derivatives thereof refers to methods that can be used to deliver an agent or composition to a desired site of biological action. These methods include, but are not limited to, parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular, intrathecal, intranasal, intravitreal, infusion, and topical injection), transmucosal injection, oral administration, suppository administration, and topical administration. Administration can be by any route, including parenteral administration. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other delivery means include, but are not limited to, the use of liposomal formulations, intravenous infusion, transplantation, and the like. One skilled in the art will appreciate other methods of administering a therapeutically effective amount of a composition of the present disclosure for preventing or alleviating one or more symptoms associated with a disease.
Disclosed herein are systems, methods, and compositions for genetically engineering cells and modulating cell behavior, e.g., by introducing exogenous polynucleotides and modulating expression of polypeptides within cells. Cells produced using the methods described herein may be suitable for therapeutically effective purposes, such as xenotransplantation, tissue regeneration, or cell-based immunotherapy. Stable expression of the exogenous polynucleotide in the primary cell can be achieved by integration of the exogenous polynucleotide into the genome of the chromosome of the cell. In general, multiple rounds of genetic manipulation may be required to produce cells with the appropriate characteristics. However, due to the intolerance exhibited by cells, it can be particularly difficult, or even impossible, to perform multiple gene targeting events on the genome of a cell over the lifetime of a single cell. One approach to circumvent this problem may be to immortalize the target cell to integrate exogenous genetic material multiple times and optionally sequentially into the cell genome. On the other hand, immortalized cells can be tumorigenic, and thus appropriate modifications to the process may be required to use the cells in vivo. Described herein are methods and compositions useful for the production of genetically engineered cells for biomedical purposes by the introduction of modulated immortalization of the cells.
System for controlling cell behavior
The present disclosure provides systems for modulating cell immortalization.
In one aspect, the present disclosure provides a system for modulating cell immortalization. The system may include: a first polynucleotide sequence comprising an immortalizing gene, wherein expression of said immortalizing gene results in the immortalization of a cell; and a second polynucleotide sequence comprising a replacement gene replacing the immortalizing gene or a fragment thereof. In some embodiments, the system may be induced by a modulator that affects the replacement or exchange between the immortalizing gene and the replacement gene such that expression of the immortalizing gene in the cell is reduced upon induction of the system with the modulator.
In some embodiments, the system can provide a platform for introducing one, two, three, four, five, six, seven, eight, nine, ten, or more gene editing events prior to immortalization. In some embodiments, the cell may undergo two or more gene editing events prior to immortalization. In some embodiments, the cell may undergo at least two gene editing events prior to immortalization.
In some embodiments, the system can provide a platform for introducing one, two, three, four, five, six, seven, eight, nine, ten, or more gene editing events after immortalization. In some embodiments, the cell may undergo two or more gene editing events after immortalization. In some embodiments, the cell may undergo at least two gene editing events after immortalization.
In some embodiments, the system can provide a platform for introducing a gene editing event after immortalization. In some embodiments, the system can provide a platform for introducing more than one gene editing event after immortalization. In some embodiments, the system can provide a platform for introducing two or more gene editing events after immortalization. In some embodiments, the system can provide a platform for introducing three or more gene editing events after immortalization. In some embodiments, the system can provide a platform for introducing four or more gene editing events after immortalization. In some embodiments, the system can provide a platform for introducing five or more gene editing events after immortalization.
In some embodiments, the system can provide a platform for introducing one or more gene editing events after immortalization reduction. In some embodiments, the system may provide a platform for introducing two or more gene editing events after immortalization reduction. In some embodiments, the system can provide a platform for introducing three or more gene editing events after immortalization reduction. In some embodiments, the system can provide a platform for introducing four or more gene editing events after immortalization reduction. In some embodiments, the system can provide a platform for introducing five or more gene editing events after immortalization reduction.
In some embodiments, the first polynucleotide sequence and the second polynucleotide sequence may be non-endogenous (e.g., heterologous). In some embodiments, the first polynucleotide sequence and the second polynucleotide sequence may be non-homologous. In some embodiments, the first polynucleotide sequence and the second polynucleotide sequence may comprise recombinant nucleic acid sequences.
First polynucleotide sequence
In some embodiments, the first polynucleotide sequence may be a DNA molecule comprising a sequence encoding an immortalizing gene. In some cases, a DNA molecule may comprise one or more coding sequences, and transcription of the DNA molecule into one or more RNA molecules may be required to induce cell immortalization. In some examples, it may be desirable to translate one or more RNA molecules into one or more polypeptides to induce immortalization. In some embodiments, the first polynucleotide comprising an immortalizing gene can be an RNA molecule. In some embodiments, the immortalizing gene is present in the cell (e.g., in the genome of the cell), and regardless of its expression, may be sufficient to induce cell immortalization.
In some embodiments, an example of an immortalizing gene can be any one of the following: polyomavirus T antigen, adenovirus E1 and E1B gene products, papillomavirus E6 and E7 proteins, hepatotrophic virus X protein, Epstein-Barr virus EBNA, LMP, BALF.1. protein, BARF1 protein, BCRF1 protein and BART gene product, herpesvirus K1 protein associated with Kaposi sarcoma virus, K2 protein, K15 protein, ORF72 product, ORF74 product, LANA and carbophil, herpesvirus saimiri STP, TI P, ORF13, ORF72, ORF73 and ORF74 product, human T cell leukemia virus type 1 Tax and HBZ protein, hepatitis C virus core protein, NS3 and NS5A protein, dominant form of Cip/Kip, Ink4, P53 and Rb family members, constitutively active form of cyclin, CDK and 2, polycomb family proteins (e.g. Bbmi b 56, autophagy protein, Bbmi protein, and apop protein), anti-necrosis factor b protein, such as Bbmi protein, Bbmi protein, Bcl/c 5, pro-protein, pro-apoptosis protein, such as, ILP2, livin/MLIAP, NIAP, survivin, Al, Bcl-2, Bcl2-L-10, Bcl-B, Bcl-W, BC1-XL, Bfl-1, BOO/DIVA, Mcl-1, NR-13, vBcl-2, vIAP, C-FLIP, V-FLIP, adenovirus E3-6.7k protein, RIDa, RID6, E3-14.7k protein, African swine fever virus A179L [5-HL ] protein, baculovirus p35, cytomegalovirus 62.7, pUL37XL [ vMIA ], UL36[ vICA ], vIRA, Epstein-Barr virus BHRF1 protein, fowl pox virus FPV039 protein, gamma herpes virus 68M8[ vMAP ] protein, hepatitis C6, NS2, BHNS 5, herpes virus BHRF1 protein, BCP 27, BCH 27H 27, BCH 27H 11, BCH 27, BCH 3, BCH, K7 protein, K13 protein, KSBcl-2, myxoma virus M11L, parapoxvirus ORFV125, vaccinia virus CrmaA/SPI-2, F1L, NIL, SPI-1), retroviral oncoproteins (e.g., V-Abl, V-Akt, V-Crk, V-Cyclin, V-ErbA, V-Erbb, V-Fos, V-Ha-ras, V-Jun, V-Ki-ras, V-Mos, V-Myc, V-P3K, V-Sis, and V-Src), telomerase reverse transcriptase (TERT), oncogenic mutants of cellular oncogenes, tumor suppressor genes, functional variants thereof, modifications thereof, and combinations thereof.
In some embodiments, the immortalizing gene can be a simian virus (SV40) T antigen gene sequence or a Human Papilloma Virus (HPV) E6/E7 gene sequence, and introduction of these genes into the primary cell can result in cell immortalization. Other cell immortalization systems include, but are not limited to, the introduction of oncogenes, such as the Myc T58A, RasV12, p53 genes.
In some embodiments, the immortalizing gene can be telomerase reverse transcriptase protein (TERT). TERT is inactive in most somatic cells, but when TERT is exogenously expressed, the cells are able to maintain sufficient telomere length to avoid replicative senescence. The immortalization of cells by telomerase (TERT) overexpression may help to maintain a stable genotype and retain key phenotypic markers. In some embodiments, TERT can be a human TERT (htert). In some embodiments, TERT can be a non-human mammalian TERT. In some embodiments, the immortalized cell can be a non-human mammalian cell (e.g., a porcine cell), in which case the TERT gene is selectively derived from the organism from which the cell is derived.
In some embodiments, the immortalizing gene may be a Myc phosphorylation-deficient mutant T58A, S62A, or T58A/S62A. In some embodiments, the immortalizing gene can be Myc T58A.
In some embodiments, the immortalizing gene can be the SV 40T antigen.
In some embodiments, the immortalizing gene may be CDK 4.
In some embodiments, the immortalizing gene can be HOXB.
In some embodiments, the immortalizing gene may be HOXA 9.
In some embodiments, the immortalizing gene may be cMyc.
In some embodiments, the immortalizing gene can be Bmi 1.
In some embodiments, the immortalizing gene can be more than one immortalizing gene. In some cases, the immortalization genes comprise two immortalization genes, e.g., a TERT gene and a Myc T58A gene.
In some embodiments, the immortalizing gene is operably linked to a promoter. In some cases, a conditional promoter and/or inducible promoter is used to conditionally and/or inducibly express the immortalizing gene. An example of an inducible promoter may be a tetracycline-inducible promoter.
In some embodiments, expression of the immortalizing gene can trigger cell division, inhibit cell death or apoptosis, and prolong the half-life of the cell. Typically, primary cells may undergo a limited number of cell cycles, i.e. they exhibit only a limited number of cell cycles. In some cases, for example after passage 2, 3, 4, 5 or 6, primary cells can slow down cell division and enter the senescence stage, where they eventually undergo apoptosis and die. In some embodiments, introduction of an immortalizing gene into a cell can greatly increase the number of times a cell comprising the immortalizing gene undergoes a cell cycle and is passaged in vitro.
In some embodiments, the first polynucleotide sequence comprising the immortalizing gene can be integrated into the genome of the cell. The immortalizing gene may be integrated into the chromosome of the cell. Alternatively, or in addition, the immortalizing gene may be integrated in the mitochondrial DNA. In another alternative, the immortalizing gene may not be integrated into the chromosome or mitochondrial DNA. In one example, the immortalizing gene can be episomal.
Expression of the immortalizing gene can immortalize the cell. Immortalizing genes can trigger cell division, inhibit cell death or apoptosis, and prolong the half-life of cells. In some cases, expression of the immortalizing gene increases the number of cell divisions of the cell by at least 2-fold as compared to the same cell without the immortalizing gene. In some embodiments, expression of the immortalizing gene increases the number of cell divisions by at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1000-fold, or more, as compared to the same cell that does not express the immortalizing gene. In some cases, the cell cycle of a cell expressing an immortalizing gene can be increased at least 2-fold compared to the same cell without the immortalizing gene. In some cases, a cell expressing an immortalizing gene can exhibit an at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1000-fold increase in lifespan as compared to the same cell that does not express the immortalizing gene.
In some embodiments, the first polynucleotide sequence is integrated at a specific site in the cell. In some cases, the immortalizing gene can be integrated at the AAVS1 locus in the genome of the cell. In some cases, the immortalizing gene can integrate at a locus in the genome of the human cell that has the sequence of, or has at least 90% sequence identity to, a lentiviral integration site (LP) (e.g., intron 1 of BMP5 gene, intron 9 of sbp2 gene, intron 2 of Dcn gene, intron 2 of Smad6 gene, etc.). In some embodiments, the immortalizing gene may be integrated at the GGTA1 locus. In some embodiments, the immortalizing gene may be integrated at the CMAH locus. In some embodiments, the immortalizing gene may be integrated at the B4GALNT2 locus. In some embodiments, the immortalizing gene may be integrated at the B2M locus. In some embodiments, the immortalizing gene can be integrated at the ROSA26 locus. In some embodiments, the immortalizing gene may be integrated at the COLA1 locus. In some embodiments, the immortalizing gene can be integrated at the TIGRE locus.
Nucleic acid editing part
In some embodiments, the first polynucleotide sequence can be integrated into the genome of the cell by a nucleic acid editing portion comprising a nucleic acid cleavage portion. In some embodiments, the nucleic acid editing moiety can initiate cleavage of a nucleic acid strand by the nucleic acid cleavage moiety. In some embodiments, the nucleic acid editing moiety may be capable of inducing homologous recombination of DNA strands. In some embodiments, the nucleic acid editing moiety may be capable of effecting DNA repair and/or ligation. In some embodiments, the nucleic acid editing portion can comprise any one or combination of components capable of: initiating nucleic acid strand breaks, such as DNA double strand breaks; inducing homologous recombination of the DNA strands; DNA repair and/or ligation is achieved. In some embodiments, the nucleic acid editing portion can edit the target gene or target nucleic acid by a nuclease to effect knock-in (insertion), knock-out (deletion), and/or mutation of the target gene or target nucleic acid, thereby modulating expression and/or activity of the gene or nucleic acid.
Exemplary gene-editing nucleases can include Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases, also known as CRISPR/Cas system ZFNs, TALENs, and CRISPR/Cas are sometimes referred to as designer nucleases. In some cases, robust homology-directed gene targeting or knock-in can be achieved by the delivery of a binding designer nuclease-induced Double Strand Break (DSB) formation to a donor DNA template whose sequence shares identity with the region flanked by the target chromosomal lesion.
Zinc finger nucleases: in some embodiments, the gene-editing nuclease may be a zinc finger nuclease. In some embodiments, the zinc finger nuclease may comprise Cys2-His2Zinc finger domain. In some embodiments, the zinc finger may consist of approximately 30 amino acids in the conserved β β β α configuration. In some embodiments, the zinc finger proteins may have modular structures, which may be a framework of interest for designing customized DNA binding proteins. Each zinc finger domain can touch 3-4 base pairs (bps) in the major groove of DNA. In some embodiments, the zinc finger nuclease may be a fusion of a non-specific DNA cleavage domain from a fokl restriction endonuclease with a zinc finger protein. In some embodiments, the zinc finger nuclease may be an engineered protein comprising an engineered zinc finger. Engineered zinc fingers are also commercially available. In some embodiments, one class is referred to as zincZinc finger proteins that refer to nicking enzymes are useful. Zinc finger nickases contain an inactivating mutation in one of the two fokl cleavage domains. The ZFN enzyme generates only single-stranded DNA breaks and induces homology-directed repair (HDR), without activating the mutagenic non-homologous end joining (NHEJ) pathway.
Transcription activator-like effector nucleases (TALENs): in some embodiments, the gene-editing nuclease can be a TALEN, which can be a naturally occurring protein comprising a DNA binding domain consisting of 33-35 amino acid repeat domains, each repeat domain recognizing one base pair each. Like zinc fingers, modular TALE repeats join together to recognize contiguous DNA sequences. In some embodiments, improved ZFNs and TALEN heterodimers with optimal cleavage specificity and reduced toxicity can be used.
RNA-guided nuclease-CRISPR/Cas system: in some embodiments, the gene-editing nuclease may be a CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a programmable RNA-guided DNA endonuclease. In some embodiments, the CRISPR-Cas system can have multiple gene disruption capabilities. In some embodiments, the CRISPR-Cas system may comprise a trans-activating chimeric RNA (tracrrna), which is a non-coding RNA that facilitates crRNA processing and is necessary to activate RNA-guided cleavage by a Cas protein (e.g., Cas 9). In some embodiments, the Cas endonuclease may be a modified enzyme. In some embodiments, the modified Cas9 protein may comprise at least one modification that alters editing preference relative to the composition of the wild-type. In some embodiments, the editing preference may be a particular insertion or deletion within the targeted region. In some embodiments, the at least one modification increases the formation of one or more specific indels. In some embodiments, the at least one modification may be in a binding region that includes a targeting region and/or a PAM interaction region. In some embodiments, the one or more modifications are located in or near the RuvC domain. In some embodiments, the one or more modifications may be located in or near the HNH or Nuc domains. In some cases, the one or more modifications are in or near the bridge helix. In some embodiments, the Cas nuclease may be any Cas protein family member, e.g., Cas12a, Cas12c, Cas13a, Cas13b, Cas13c, Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, or Cas 10. In some cases, the CRISPR-associated nuclease may be MAD 7. In some embodiments, the CRISPR-associated nuclease may be HEPL (HEF1-Efs-p130 Cas-like). In some embodiments, the CRISPR system can be an editing system, wherein the Cas enzyme further comprises a reverse transcriptase, and wherein the guide RNA comprises two components, one fragment that binds to the nicked DNA at the target site, the other RNA fragment comprising a sequence that replaces the gene or fragment thereof. Reverse transcriptase allows the simultaneous formation of DNA with the replacement gene sequence at the nicked target DNA site.
Alternatively, in some embodiments, the nucleic acid editing moiety can be a replication initiation protein (Rep) for adenovirus-mediated gene editing.
In some embodiments, the nucleic acid editing moiety can be an integrase protein for retrovirus-mediated gene editing. In some cases, one or more integration-related mechanisms may be combined to achieve the intended purpose of target site-specific integration, thereby achieving high efficiency and reducing off-target effects. In turn, the above is directed to reducing integration-related toxicity.
In some embodiments, a first polynucleotide sequence comprising an immortalizing gene can be exchanged with a second polynucleotide sequence comprising a replacement gene. In some embodiments, the first polynucleotide sequence may be replaced entirely by the second polynucleotide sequence. In some embodiments, the sequence encoding the immortalizing gene in the first polynucleotide sequence may be replaced entirely by the second polynucleotide sequence. In some embodiments, the second polynucleotide sequence replaces a fragment of the sequence encoding the immortalizing gene. In each of the above cases, replacement of the first polynucleotide sequence or fragment thereof with the second polynucleotide sequence or fragment thereof reduces expression of the immortalizing gene.
In some embodiments, the second polynucleotide sequence comprising the replacement gene may replace at least one nucleotide of the sequence encoding the immortalizing gene in the first polynucleotide sequence such that expression of the immortalizing gene is reduced. In some cases, the second polynucleotide sequence comprising the replacement gene can replace at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides of the sequence encoding the immortalizing gene. At least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides. In some cases, the second polynucleotide sequence replaces about 10, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 nucleotides of the sequence encoding the immortalizing gene. In some cases, the second polynucleotide sequence replaces about 105, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 350, about 400, about 450, about 500, about 600, about 700, about 800, about 900, or about 1000 nucleotides of the sequence encoding the immortalizing gene. In some cases, the second polynucleotide sequence replaces more than 500, more than 600, more than 700, more than 800, more than 900, more than 1000, more than 1100, more than 1200, more than 1300, more than 1400, more than 1500, more than 1600, more than 1700, more than 1800, more than 1900, or more than 2000 nucleotides of the sequence encoding the immortalizing gene.
In some embodiments, the exchange between the immortalizing gene and the second polynucleotide sequence encoding the replacement gene reduces the expression of the immortalizing gene. In some cases, expression of the immortalizing gene is reduced by at least 2-fold compared to its expression prior to the exchange with the second polynucleotide sequence. In some cases, expression of the immortalizing gene is reduced by at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold as compared to its expression prior to the exchange with the second polynucleotide sequence. At least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times to at least 18 times, at least 19 times, at least 20 times, at least 21 times, at least 22 times, at least 23 times, at least 24 times, at least 25 times, at least 26 times, at least 27 times, at least 28 times, at least 29 times, at least 30 times, at least 31 times, at least 32 times, at least 33 times, at least 34 times, at least 35 times, at least 36 times, at least 37 times, at least 38 times, at least 39 times, at least 40 times, at least 41 times, at least 42 times, at least 43 times, at least 44 times, at least 45 times, at least 46 times, at least 47 times, at least 48 times, at least 49 times, or at least 50 times. In some cases, expression is reduced at least 55-fold, at least 60-fold, at least 65-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 85-fold, at least 90-fold, at least 95-fold, at least 100-fold, at least 105-fold, at least 110-fold, at least 115-fold, at least 120-fold, at least 125-fold, at least 130-fold, at least 135-fold, at least 140-fold, at least 145-fold, at least 150-fold, at least 155-fold, at least 160-fold, at least 165-fold, at least 170-fold, at least 175-fold, at least 180-fold, at least 185-fold, at least 190-fold, at least 200-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, as compared to expression prior to the exchange of the immortalizing gene with the second polynucleotide sequence, At least 47 times, at least 48 times, at least 49 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times.
In some cases, expression of the immortalizing gene is reduced by up to 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, or up to 1000-fold compared to its expression prior to the exchange with the second polynucleotide sequence.
Alternatively, or in addition, cell immortalization may involve treating the cells with one or more exogenous factors. The one or more exogenous factors can include a nucleic acid, a polynucleotide, an amino acid, a polypeptide, a lipid, a carbohydrate, a small molecule, an enzyme, a ribosome, a proteasome, a variant thereof, or any combination thereof. In some embodiments, the one or more exogenous factors can be a polynucleotide or polypeptide encoded by any immortalizing gene of the presently disclosed subject matter.
In some embodiments, expression of the immortalizing gene can be attenuated by exchanging with a second polynucleotide sequence. In some embodiments, the reduction in expression of the immortalizing gene following exchange with the second polynucleotide sequence is independent of whether the replacement gene is expressed.
Second polynucleotide sequence
In some embodiments, the second polynucleotide sequence may be a DNA molecule comprising a sequence encoding a replacement gene (e.g., a gene encoding a human protein or polypeptide of interest, rather than an immortalized gene). In some embodiments, a DNA molecule may comprise one or more coding sequences. In some embodiments, the immortalizing gene can be such an RNA molecule. In some embodiments, the immortalizing gene is present in the cell (e.g., in the genome of the cell), and regardless of its expression, may be sufficient to induce cell immortalization.
The second polynucleotide sequence comprising the replacement gene may comprise any gene encoding a protein or polypeptide. In some embodiments, the replacement gene may be a human gene. In some embodiments, the replacement gene may encode a human protein. In some embodiments, the second polynucleotide encodes a protein that enhances the allogeneic or xenogeneic compatibility of the cell. In some embodiments, the second polynucleotide expression may be stably expressed. In some embodiments, the second polynucleotide expression may be permanent expression. In some embodiments, the engineered cells may be suitable for xenotransplantation. In some embodiments, the engineered cells may be suitable for allogeneic transplantation, e.g., transfer to another person. In some embodiments, the second polynucleotide may be stably expressed. In some embodiments, the second polynucleotide may be expressed throughout the life of the cell.
For example, porcine xenografts are widely compatible with the small size and physiology of human organs and are ethically accepted by the general population of the united states. However, xenografted porcine tissue triggers a complex series of events leading to graft rejection, including: hyperacute rejection due to the presence of preformed antibodies against porcine antigens, complement activation and hypercoagulability, and an increase in innate and adaptive immune responses due to molecular incompatibility. Thus, cells can be designed to exhibit reduced expression of allogeneic or xenogeneic incompatible epitopes, and/or to induce suppression of an induced host immune response. As described in this section, a second polynucleotide sequence encoding a replacement gene may be used for this purpose.
In some embodiments, the replacement gene is capable of enhancing allogenic or xenogenic compatibility of the cell. Allogenic or xenogenic compatibility of, for example, a human host can be assessed, for example, by reduced binding of human antibodies, human complement, human MHC receptors, or human T cells, or reduced activity of mixed lymphocyte responses.
In some embodiments, the replacement gene encodes a protein that can cause enhanced immune compatibility, including one or more complement response genes (interchangeably referred to herein as complement toxicity genes), coagulation response genes (interchangeably referred to herein as coagulation genes), inflammation response genes (interchangeably referred to herein as apoptosis/inflammation genes), immune response genes (interchangeably referred to herein as cytotoxic genes), and/or immune regulatory genes.
In some embodiments, the second polynucleotide sequence comprising the replacement gene comprises one or more transgenes selected from the group consisting of an inflammatory response gene, an immune response gene, an immunomodulatory gene, and combinations thereof.
In some embodiments, the replacement gene comprises a transgene selected from the group consisting of tumor necrosis factor alpha-induced protein 3(a20), heme oxygenase (HO-1 or HMOX1), cluster of differentiation 47(CD47), and combinations thereof.
In some embodiments, the replacement gene comprises an immune response transgene selected from the group consisting of human leukocyte antigen-E (HLA-E), beta-2 microglobulin (B2M), and combinations thereof.
In some embodiments, the replacement gene comprises an immunomodulator transgene selected from programmed death ligand 1(PDL1), Fas ligand (FasL), and a combination thereof.
In some embodiments, the replacement gene comprises a coagulation responsive transgene selected from the group consisting of cluster of differentiation 39(CD39), thrombomodulin (THBD, TBM or TM), Tissue Factor Pathway Inhibitor (TFPI), and combinations thereof.
In some embodiments, the replacement gene comprises a sequence selected from the group consisting of human membrane cofactor protein (hCD46 or simply CD 46); complement response transgenes of human complement decay accelerating factor (hCD55 or CD55 for short), human MAC inhibitor factor (hCD59 or CD59 for short), and combinations thereof.
In some embodiments, the replacement gene encodes a human copy selected from the group consisting of: B2M, HLA-E, CD47, PDL1, CD46, CD55, CD59, A20, THBD, TFPI, CD39, EPCR, FASL, CIITA, CTLA-Ig and HO-1.
In some embodiments, the engineered cell expressing the replacement gene may be incapable of expressing a functional copy of an incompatible allogeneic or xenogeneic epitope.
In some embodiments, functional copies of incompatible allogeneic or xenogeneic epitopes may be deleted, mutated, or exchanged. In some cases, the incompatible allogeneic or xenogeneic epitope is selected from: GGTA, CMAH, and B4 Gal. In some cases, functional copies of incompatible allogeneic or xenogeneic epitopes (such as GGTA, CMAH, and B4Gal) are deleted, mutated, or exchanged through a system comprising the activity of a nucleic acid cleavage moiety (e.g., a nuclease), such as a CRISPR-Cas system. In some cases, the functional copies of the incompatible allogeneic or xenogeneic epitope are deleted, mutated, or exchanged by the same nucleic acid editing moiety that integrates the first polynucleotide sequence into the genome.
In some embodiments, expression of the replacement gene results in a decrease in the host's immune response to the allogeneic or xenogeneic epitope of the engineered cell. In some cases, expression of the replacement gene results in a decrease in T cell response of the host to an allogeneic or xenogeneic epitope of the engineered cell, as determined by one or more of the following exemplary results: proliferation and activation of CD8+ T cells; release cytokines such as interferon gamma (IFN- γ); production of interleukins in vivo. In some cases, expression of the replacement gene results in a decreased T cell response of the host to an allogeneic or xenogeneic epitope of the engineered cell, as determined by one or more of the following in vitro exemplary assays: tetramer assays, cytokine (IFN-. gamma.and/or IL-2) release assays, and the like.
In some embodiments, the systems described herein include a mechanism to facilitate efficient exchange of a first polynucleotide sequence or fragment thereof with a second polynucleotide sequence. The mechanism may involve a modulator to facilitate one or more exchanges, including excision, removal, and replacement of the first polynucleotide sequence with the second polynucleotide sequence. Exemplary excision of the DNA may involve restriction enzyme-mediated digestion at a restriction enzyme recognition site, which may be encoded by a region flanking the first polynucleotide sequence encoding the region of the immortalizing gene. However, other effective mechanisms as exemplified herein may be employed.
In one aspect, two or more genetic modifications can be performed on a cell to knock out a first gene and knock in a second gene. In some cases, the first gene may encode an allogeneic or xenogeneic epitope or fragment thereof with respect to the organism into which the cell is delivered. In some cases, the second gene is a transgene.
In some embodiments, the allogeneic or xenogeneic epitope may be GGTA or CMAH or B4 GAL. In some cases, GGTAs may be incompatible epitopes, and GGTAs are knocked out by genetic modification events. Alternatively, in some cases CMAH may be an incompatible epitope and knocked out by genetic modification events. In some cases, B4GAL may be an incompatible epitope, and B4GAL may be knocked out by a genetic modification event. In some cases, more than one gene selected from GGTA, CMAH, and B4GAL may be an allogeneic or xenogeneic epitope. In some cases, more than one gene selected from GGTA, CMAH, and B4GAL (which are allogeneic or xenogeneic epitopes) is knocked out by one or more genetic modification events. In some cases, knocking out one or two or three incompatible epitopes reduces the immunogenic response of an organism to the cell undergoing the genetic modification event (when introduced into the organism).
In some embodiments, the second gene may be a gene encoding a polypeptide capable of enhancing the allogeneic or xenogeneic compatibility of the cell. The second gene may encode a human protein. The second gene may encode a human polypeptide selected from B2M, HLAE, CD47, PDL1, CD46, CD55, CD59, A20, THBD, TFPI, CD39, EPCR, FASL, CIITA, CTLA-Ig and HO-1.
In some embodiments, the genetic modification event can be performed by a nuclease. One or more genetic events can be induced by one or more effector moieties comprising a nuclease. In some cases, one or more actuator portions are capable of performing two or more genetic modification events.
In some embodiments, the immortalizing gene is inactivated by expression of a gene knocked-in by a genetic modification event.
Modulators and activators
In some embodiments, the system may include recombination-mediated exchange. In some cases, the first polynucleotide sequence is flanked by a first recombinase site and the second polynucleotide sequence is flanked by a second recombinase site, and wherein the modulator comprises a recombinase capable of causing an exchange between the first polynucleotide sequence comprising, for example, an immortalized gene and the second polynucleotide comprising, for example, a replacement gene. The first recombinase site may comprise a pair of recombinase sites flanked by 5 'and 3' sites of an immortalized gene. The second recombinase site may comprise a pair of recombinase sites flanked by 5 'and 3' sites of a replacement gene. In some cases, the first recombinase site and the second recombinase site are sites that bind the same recombinase. The first recombinase site and the second recombinase site are sites oriented in the same 5 'to 3' direction.
In some cases, the system may comprise one or more modulators. Exemplary modulators may include, but are not limited to, recombinases, ribozymes having recombinase activity, transposases, or integrases. In some cases, the system can comprise a third polynucleotide sequence encoding a modulator. Induction and/or activation of the modulator affects the exchange between the immortalizing gene and the replacement gene. The modulator may be an enzyme. In some cases, the system comprises a third polynucleotide sequence comprising a sequence encoding a modulator. In some cases, the third polynucleotide sequence encoding the modulator may be constitutively expressed or may be expressed under a cell-specific or tissue-specific promoter, or may be manipulated under an inducible promoter.
In some cases, the modulator can be a recombinase. In some cases, the recombinase may be a sequence-specific recombinase. In some cases, the recombinase may be a site-specific recombinase. In some cases, the recombinase may be selected from: cre recombinase, Flp recombinase, Hin recombinase, Tre recombinase and PhiC31 recombinase (φ C31).
In some cases, the modulator may comprise a ribozyme having recombinase activity. Ribozymes having recombinase activity may be selected from the group consisting of GIR1 branching ribozymes, glmS ribozymes, class I self-splicing introns, class II self-splicing introns, spliceosomes, hairpin ribozymes, hammerhead ribozymes, HDV ribozymes, rRNA, RNase P, Twister ribozymes, Twister sister ribozymes, VS ribozymes, Pistol ribozymes, and Hatchet ribozymes, or variants thereof.
In some cases, the modulator may be a Cre recombinase. For example, in the above-described regulatable expression system, the first and second polynucleotide sequences may be flanked by loxP sites, respectively, each located at either end (5 '-and 3' -ends), flanked by each polynucleotide sequence, respectively. The loxP sites form recognition sites for Cre recombinase, which induces homologous recombination-mediated excision of the lox sites and exchange of the first and second polynucleotide sequences. Additional or alternative lox sites can be selected from those known in the art, including naturally occurring loxB, loxL, and loxR, as well as a number of mutant or variant lox sites, such as loxP511, loxP514, lox Δ 86, lox Δ 117, loxC2, loxP2, loxP3, and lox P23.
Alternatively, the modulator may be FLP recombinase. For example, in the regulatable expression system described above, the first and second polynucleotide sequences may be flanked by "frt sites", respectively. As used herein, frt site refers to a nucleotide sequence of the FLP gene product of a yeast 2 micron plasmid (FLP recombinase) that can catalyze site-specific recombination.
In some embodiments, the modulator may be a PhiC31 recombinase. For example, in the regulatable expression system described above, the first and second polynucleotide sequences may be flanked by "attP" and "attB" sites, respectively, or any variant thereof which improves site-specific recombination by the enzyme PhiC31 recombinase.
In one aspect, the modulator may be inducible. For example, the Cre recombinase may be inducible. In one embodiment, the modulator may be a fusion protein. In some cases, the modulator may be part of a fusion protein comprising at least one activator-binding domain.
In some cases, the activator-binding domain can be a ligand-binding domain of an Estrogen Receptor (ER).
In some cases, the activator can be a synthetic peptide. In some embodiments, the activator may be tamoxifen. Tamoxifen inducible systems may comprise a reversible switch that may provide reversible control over transcription of one or more genes regulated by the system. Particularly when used in conjunction with the Cre/Lox recombinase system, the tamoxifen/estrogen receptor modulatory system allows for spatiotemporal control of gene expression, in which Cre recombinase fuses with mutated forms of the human estrogen receptor ligand binding domain to produce tamoxifen-dependent Cre recombinase.
In some embodiments, the modulator sequence encoded by the third polynucleotide sequence may be capable of being activated upon induction of the system with an activator. The third polynucleotide sequence encoding the regulator sequence may be operably linked to a promoter. In some embodiments, the promoter may be regulated by an activator or repressor. Any inducible or repressible promoter can be used. Exemplary promoters that can be modulated include, but are not limited to, the Tet-on and Tet-off promoters inducible (Tet-on) or repressible (Tet-off) by tetracycline.
In one aspect, the present disclosure provides a system for genetically modifying a cell. The system may comprise a polynucleotide sequence comprising an immortalizing gene, wherein expression of the immortalizing gene causes immortalization of the cell and the immortalizing gene is inactivated by an inactivating moiety. The system can comprise a nucleic acid editing moiety comprising a nucleic acid cleavage moiety. In some cases, the system can further comprise an inactivating moiety comprising a nucleic acid editing moiety having a nucleic acid cleaving moiety. The induction of the inactivating moiety inactivates expression of the immortalizing gene in the cell.
In some embodiments, the nucleic acid editing moiety may be capable of inducing at least one genetic modification in a cell. In some embodiments, the nucleic acid editing moiety can be configured to complex with a target polynucleotide in a cell to induce at least one genetic modification in the cell. The nucleic acid editing moiety may be capable of inducing at least one genetic modification in the cell such that the at least one genetic modification enhances allogeneic or xenogeneic compatibility of the cell.
The genetic modification may be a germline modification.
In one embodiment, the inactivating moiety may comprise a recombinase.
In some embodiments, the polynucleotide sequence may be chromosomal.
In some embodiments, the sequence encoding the immortalizing gene may be flanked by recombinase sites and may be acted upon by a recombinase capable of deleting the immortalizing gene.
In some embodiments, the inactivating moiety may comprise a recombinase that is capable of inverting the immortalized gene flanked by recombinase sites from an active 5'-3' orientation to an inactive 3'-5' orientation and recombining such that the gene can no longer be transcribed. In some cases, the immortalized gene or fragment thereof can be flanked by more than one pair of recombinase sites. In some cases, the flanking pairs of recombinase sites may constitute a first pair of recombinase sites, and the action of the first recombinase on the first pair of recombinase sites affects the inversion of the immortalized gene or fragment thereof. In some cases, the recombinase sites comprise LoxP sites and Lox2272 sites in alternating orientations on either side of the polynucleotide sequence, and wherein the recombinase is Cre recombinase.
In some embodiments, the inactivating moiety comprises an additional nucleic acid editing moiety. The additional nucleic acid editing moiety may be capable of removing the immortalizing gene from the polynucleotide sequence to inactivate expression of the immortalizing gene in the cell.
In some embodiments, the system can further comprise an additional polynucleotide sequence comprising a replacement gene flanked by a pair of recombinase sites that can be different from the first pair of recombinase sites and that are capable of being acted upon by a second recombinase; and the immortalization gene is flanked by additional recombinase sites enabling the second recombinase to cause an exchange of the immortalization gene with the replacement gene. In some cases, the second recombinase can be a Flp recombinase and the respective other pair of recombinase sites are FRT sites. In some cases, the second recombinase may be a Hin recombinase or a Tre recombinase, and the respective additional pairs of recombinase sites are compatible with the recombinase sites.
In some embodiments, the polynucleotide sequence encoding the immortalizing gene may further comprise a recombinase site and the immortalizing gene, which may be flanked by a promoter. In some embodiments, the recombinase site may be flanked at the 5 'end by a promoter of the immortalized gene, and the coding sequence of the immortalized gene is flanked at the 3' end; the inactivating moiety comprises a recombinase capable of inserting a termination sequence between recombinase sites. In some embodiments, the position of the recombinase site may be positioned such that it flanks the immortalized gene and the replacement gene may comprise a copy of the immortalized gene or a fragment thereof, with the termination sequence embedded within the coding region of the immortalized gene.
In some embodiments, the inactivating moiety may comprise a site-specific recombinase. In some cases, the site-specific recombinase may be a CRISPR-associated recombinase, or a transposase. In some embodiments, the inactivating moiety may be a site-specific recombinase that can delete the polynucleotide sequence encoding the immortalizing gene or fragment thereof. In some embodiments, the inactivating moiety may be a site-specific recombinase that introduces a termination sequence within the immortalized gene such that the immortalized gene is not transcribed or translated, or does not produce an active protein. In some cases, the site-specific recombinase may replace the nucleotide comprising the start codon of the immortalized gene with a termination sequence.
In some embodiments, the inactivating moiety may insert a termination sequence 5 'or 3' to the start codon of the immortalized gene such that the immortalized gene is not transcribed or translated, or does not produce an active protein.
In some embodiments, the termination sequence may comprise a stop codon. In some cases, the termination sequence can comprise a stop codon and a polya signal sequence. In some embodiments, the termination sequence may comprise a nucleotide sequence that forms a series of nonsense codons that can undergo chain termination. In some embodiments, the termination sequence may comprise one copy of the termination sequence. In some embodiments, two or three or more copies of the termination sequence can be in tandem. In some embodiments, two or three or more termination sequences may be in altered reading frames.
In some embodiments, a nucleic acid editing moiety comprising a nucleic acid cleavage moiety may be capable of inducing at least one genetic modification in a cell. In some embodiments, the genetic modification may comprise deletion of the polynucleotide sequence. In some embodiments, the genetic modification may comprise insertion of a polynucleotide sequence. In some embodiments, the genetic modification may comprise a substitution of a polynucleotide sequence. In some embodiments, the genetic modification may comprise any combination of deletion, insertion, and substitution of the polynucleotide sequence. In some embodiments, the genetic modification may comprise a deletion of the immortalizing gene or a fragment thereof, wherein expression of the immortalizing gene is reduced or attenuated. In some embodiments, the genetic modification alters the immortalizing gene, thereby reducing expression of the immortalizing gene. In some embodiments, the genetic modification alters the immortalizing gene such that no functional protein is translated from the immortalizing gene.
In some embodiments, the polynucleotide sequence comprising the immortalizing gene is chromosomal. In some embodiments, the polynucleotide sequence comprising the replacement gene may be integrated in the chromosome. In some embodiments, the polynucleotide sequence may be integrated at the AAVS1 locus in the genome of the cell. In some embodiments, the nucleic acid editing moiety is capable of integrating the polynucleotide sequence at the AAVS1 locus in the genome of the cell. In some embodiments, the immortalizing gene may be integrated at the GGTA1 locus. In some embodiments, the immortalizing gene may be integrated at the CMAH site. In some embodiments, the immortalizing gene may be integrated at the B4GALNT2 locus. In some embodiments, the immortalizing gene may be integrated at the B2M site. In some embodiments, the immortalizing gene can be integrated at the ROSA26 locus. In some embodiments, the immortalizing gene may be integrated at the COLA1 locus. In some embodiments, the immortalizing gene can be integrated at the TIGRE locus.
In some embodiments, the genetic modification may comprise deletion or knock-out of a gene or fragment thereof encoding an incompatible allogeneic or xenogeneic epitope relative to the organism into which the cell may be introduced. In some cases, the incompatible allogeneic or xenogeneic epitope may comprise GGTA, CMAH, or B4 Gal. As deemed appropriate by those skilled in the art, any alternative or additional allogeneic or xenogeneic epitope may be targeted to the deletion, depending on the source of the cell and the organism into which the cell may be introduced.
In some embodiments, the genetic modification may comprise insertion or addition or knock-in of a gene or fragment thereof encoding a human copy of the gene, which may be B2M, HLAE, CD47, PDL1, CD46, CD55, CD59, a20, THBD, TFP1, CD39, EPCR, FASL, CIITA, CTLA-Ig, or HO-1. Any alternative or additional gene may be considered for insertion as deemed appropriate by the person skilled in the art. The addition or deletion of one or more genes by genetic modification described herein can be used to increase the allogeneic or xenogeneic compatibility of cells for cell or tissue treatment purposes.
In some embodiments, genetic modification may include multiple modification events, such as knocking out one gene and knocking in another gene.
In some embodiments, genetic modification may include knocking out one gene, and knocking in more than one gene. The knock-out and knock-in events can occur simultaneously or consecutively. Knock-in genes may include, for example, immortalizing genes, regulatory genes, recombinase genes, or any gene suitable for the purpose.
In some embodiments, the system comprises one or more additional polynucleotide sequences encoding additional recombinase enzymes.
In some cases, the recombinase may be capable of being activated upon induction of the system with an activator. In some cases, the recombinase may be part of the fusion protein. In some cases, the fusion protein further comprises at least one activator-binding domain, e.g., the activator-binding domain can be a ligand-binding domain of an Estrogen Receptor (ER). In some cases, the activator may be tamoxifen.
In some embodiments, the inactivating moiety may be a polynucleotide sequence encoding a recombinase enzyme. In some cases, the polynucleotide sequence encoding the recombinase may be operably linked to a promoter.
In some embodiments, the promoter may be regulated by an activator or repressor. Any inducible or repressible promoter can be used. In some embodiments, the promoter may be inducible. In some cases, the promoter may be activated by an activator. In some embodiments, the polynucleotide sequence encoding the recombinase may be operably linked to an inducible promoter system that is regulated by the activator.
In some embodiments, the inducible promoter system may comprise a tetracycline-inducible system.
In some embodiments, the recombinase may be operably linked to a tet-repressible promoter system.
In some embodiments, the activator can be a tetracycline.
In one embodiment, the system described herein is induced by an activator which activates a modulator, preferably inside a cell expressing a transgene comprising a polynucleotide comprising an immortalizing gene, inducing a reduction or attenuation of the expression of the immortalizing gene. In some embodiments, the reduction of the immortalizing gene may be 1% to 100% of the level of expression prior to the induction system, or about 1% to 99%, or about 5% to 90%, or about 10% to 80%, or about 20% to 70%, or about 30% to 60%, or about 40% to 50%, or about 1% to 5%, 1% to 10%, 1% to 20%, 1% to 30%, 1% to 40%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, or about 1% to 95% of the level of expression prior to the induction system. In some embodiments, the reduction in immortalizing genes may be about 100% of the expression level prior to induction of the system.
Vectors and constructs
The polynucleotide sequences described herein can be introduced into a cell via a vector. Vectors known in the art that can be used in the present invention include viral vectors and episomal nucleic acids, e.g., plasmids or nucleic acid fragments. The vector may be a viral vector. Depending on the purpose, the carrier may comprise one or more nanoparticles, lipids, and any other suitable components for delivering nucleic acids within a cell. The vector may be used for transient, long-term or stable expression of the polynucleotide sequence. Vectors that integrate genetic material into the genome of the cell may be used. Vectors capable of integrating the polynucleotide sequence into the chromosome of the cell may be used. Suitable vectors include viral vectors, such as lentiviral vectors, or certain adeno-associated viral vectors (AAV). Vectors that integrate the polynucleotide sequence into the extrachromosomal genetic material of the cell can also be used.
In some embodiments, the polynucleotide sequence may also be contained within a suitable vector comprising one or more lipids. In some embodiments, the carrier comprising one or more lipids may comprise at least one cationic lipid. In some cases, the lipids can form liposomes.
In some embodiments, the polynucleotide sequence may be delivered in association with one or more peptides. Peptides may comprise short oligopeptides comprising 5-50 amino acids. In some embodiments, the polynucleotide sequence may be targeted to a particular cell or tissue by a vector comprising a target protein (e.g., an antibody).
In one aspect, the first polynucleotide sequence comprising the immortalizing gene is comprised in a suitable nucleic acid vector. In another aspect, the second polynucleotide sequence comprising the replacement gene is contained in a suitable nucleic acid vector. In some cases, the vector may be a viral vector or a plasmid vector. Viral vectors useful for the purposes described herein can be any one of a retroviral family based vectors including alpharetroviruses, betaretroviruses, delta retroviruses, epsilon retroviruses, gamma retroviruses, lentiviruses and foamy virus genera, parvoviridae, hepatoviridae, herpesviridae (e.g., Epstein-Barr virus and human herpesvirus 6), papillomaviruses, polyomaviridae (e.g., SV40 and Merkel cell polyomaviruses), adenoviridae, baculoviridae, circoviridae, and poxviridae), and chimeric viral vectors containing two or more different viral elements. The viral vector is a replication-defective viral vector, such as a lentivirus vector having the ability to integrate. Suitable viral vectors are available from, inter alia, human immunodeficiency virus (HTV), simian immunodeficiency virus (STY), equine immunodeficiency virus (EIAV), Bovine Immunodeficiency Virus (BIV), Caprine Arthritis Encephalitis Virus (CAEV), visna virus, jembara disease virus (JOY) and feline immunodeficiency virus (FTV). In another embodiment, the vector is a non-viral integration-competent vector based on a transposase, integrase or nuclease and appropriate templates for transposition, integration and homologous recombination, respectively. For an overview of suitable carrier systems, reference is made to Woodard and Calos, 20.1.5, which are incorporated herein by reference.
The vectors, preferably lentiviral vectors or other retroviral vectors of the present disclosure, may also comprise other elements, such as Long Terminal Repeat (LTR), transactivation response (TAR) elements, Primer Activation Signal (PAS), Primer Binding Site (PBS), packaging signal, Rev Response Element (RRE), Constitutive Transport Element (CTE), RNA Transport Element (RTE), Central Terminal Sequence (CTS), central polypiperidic region (cPPT), polypurine region (PPT), post-transcriptional regulatory elements such as human hepatitis b virus (HVBPRE) or Woodchuck Hepatitis Virus (WHVPRE), Internal Ribosome Entry Site (IRES) or 2A peptide-like sequences, and small drug regulatory repressor proteins, such as tetracycline (Tc) -controlled hybrid proteins rtTR-KRAB and CymR repressor.
The LTRs contain repetitive sequences important for transcription of the retroviral genome (e.g., TAR elements), reverse transcription (e.g., PAS), and host chromosomal DNA integration. Reverse transcription of retroviral genomes is also typically dependent on PBS and PPT, while reverse transcription of lentiviral and lentiviral vector genomes also requires CTS and cPFr. CTS and cPPT also play a role in nuclear import into retroviral genomes. In some cases, the packaging signal directs the introduction of the retroviral genome into the viral particle.
In some embodiments, the RRE is important for efficient nucleoplasmic export encoded by lentiviral and lentiviral vectors, non-spliced and incomplete splicing. Since retroviral packaging occurs in the cytoplasm, RNA, including the lentiviral genome, is critical for the production of viral progeny. CTE and RTE have similar effects.
In some embodiments, post-transcriptional regulatory elements of the hepadnaviridae family, as well as RREs, CTEs, and RTEs of the retroviridae family, may be introduced into the vector, which may help increase expression of under-expressed genes or polynucleotide sequences. Insertion of such elements in the untranslated region (UTR) of the coding sequence of a lentiviral or other retroviral vector can increase its expression level.
In some embodiments, the IRES is included in a suitable region of the vector to separate the expression of the two polypeptides from a single polynucleotide construct. Alternatively, or in addition, a sequence encoding a peptide 2A (-like) peptide can be introduced that separates the coding sequences for the two polypeptides from a single transcript, for self-cleavage upon protein translation.
In some cases, the first polynucleotide sequence and the same polynucleotide sequence may be comprised in the same polynucleotide molecule. In some cases, the first polynucleotide sequence and the second polynucleotide sequence may be part of a single transcript, wherein the second polynucleotide sequence may be interrupted by a gene flanked by loxP sites comprising the first polynucleotide sequence flanked by loxP sites.
In some embodiments, the two polynucleotide sequences are contained in two separate vectors.
In some embodiments, the two polynucleotide sequences are contained in the same vector, e.g., a viral AAV vector. In some embodiments, the vector may be capable of inserting the polynucleotide sequence into a specific locus within a chromosome. In some embodiments, the locus is AAVS 1.
In some embodiments, the two polynucleotide sequences may be contained in separate distinct vectors.
In some cases, the second polynucleotide sequence may be comprised in a prokaryotic plasmid vector, such as a shuttle vector. In some cases, the second polynucleotide sequence can be comprised in a prokaryotic plasmid vector.
In some embodiments, the first polynucleotide sequence and/or the second polynucleotide sequence comprises a protein coding region operably linked to a promoter. In some cases, the immortalizing gene in the first polynucleotide sequence is operably linked to a promoter. Suitable promoters may be heterologous promoters. Suitable promoters may be homologous promoters. Suitable promoters may be cell or tissue specific promoters. Suitable promoters may be inducible promoters. Exemplary promoters that can be used in the constructs described in this section can include, but are not limited to: EF1 alpha (human elongation factor 1 alpha) promoter, Cytomegalovirus (CMV) promoter, hybrid mammalian promoter (CAG), phosphoglycerate kinase (PGK) promoter, human nuclear promoter U6, methionine-inducible promoter, tetracycline responsive element promoter (TRE), tamoxifen (ER)TAM) Response promoter, human keratin 14 promoter, human α 11 integrin promoter, Fibroblast Activation Protein (FAP) promoter, procollagen α 1(Col1a1) promoter, porcine fatty acid binding protein (aP2) promoter, calcium-calmodulin-dependent protein kinase II promoter (CaMKIIa), alcohol dehydrogenase promoter, platelet-derived growth factor- β (PDGFR β) promoter, fibroblast-specific promoter (FSP1), mammalian Uteroglobin (UG) vector, actin promoter (Ac), and the like. In some cases, the intermediate promoter used in the cloning process can be a bacterial promoter, such as the T7, Sp6, araBAD, trp, Ptac, and pL promoters. Suitable promoters may be viral promoters. Exemplary viral promoters may include, but are not limited to: polyhedrin promoter (PhH) of Baculaviridae, enhancer of CMV and immediate early promoter(P CMV), the early promoter of SV40 (P SV40), the thymidine kinase promoter of HSV 1(P TK) or the 5' LTR promoter of HIV (P LTR).
In some embodiments, constitutive promoters are used to express one or more polynucleotide sequences, e.g., EF1 α, CMV, CAG, and the like.
In some embodiments, inducible promoters are used to express one or more polynucleotide sequences, for example, a tetracycline responsive element promoter (TRE) or tamoxifen (ER)TAM) Responsive promoters, and the like.
In some embodiments, tissue-specific or cell-specific promoters are used to express one or more polynucleotide sequences, e.g., for fibroblast-directed expression, the following exemplary promoters are contemplated; fibroblast specific promoter (FSP1), human α 11 integrin promoter, Fibroblast Activation Protein (FAP) promoter, procollagen α 1(Col1a1) promoter, and the like.
In some embodiments, the immortalizing gene may comprise a termination sequence at the 3' end and in frame with the reading sequence of the coding region of the immortalizing gene. In some cases, the termination sequence is flanked on the 3' end by the coding sequence of the immortalizing gene. Exemplary termination sequences can be poly a sequences of various origins, such as BGH poly a sequences, HSV tk poly a sequences, hGH poly a sequences, SV40 termination sequences, rbGlob poly a sequences, woodchuck multiple transcription regulatory element (WPRE) poly a sequences.
In some embodiments, the first polynucleotide sequence further comprises a reporter sequence upstream or downstream of the sequence encoding the immortalizing gene, such that the reporter sequence is operably linked to a promoter that drives expression of the immortalizing gene. The sequence encoding the reporter gene may be positioned such that it is in frame with the nucleotide sequence encoding the immortalizing gene and is a biological marker for expression of the gene flanked by loxP, wherein said gene is not removed or replaced by the activity of Cre recombinase. In some cases, the sequence encoding the immortalizing gene is operably linked to a promoter at the 5 'end, in-frame with the nucleotide sequence encoding the reporter gene downstream of the promoter, and in-frame with the termination sequence at the 3' -end of the reporter gene.
In some embodiments, the sequences encoding the immortalizing gene and the reporter gene are linked by a sequence encoding a protease cleavage sequence. In some cases, the protease cleavage sequence can be an endogenous protease cleavage sequence. In some cases, the protease cleavage sequence is a T2A sequence.
In some embodiments, the reporter gene may encode a protein that is readily detectable and is a marker of immortalizing gene expression. The protein is also a marker for successful removal of the gene flanked by loxP when Cre is expressed or activated. In some cases, the reporter gene encodes a fluorescent protein, such as Green Fluorescent Protein (GFP), Red Fluorescent Protein (RFP), Yellow Fluorescent Protein (YFP), luciferase (e.g., Renella luciferase, firefly luciferase, Gaussia luciferase), and the like.
Method for controlling cell behavior
The present disclosure provides methods of introducing or using any of the subject systems for controlling cell behavior as described in the present disclosure. In one aspect, the present disclosure provides a method for modulating cell immortalization. The method may comprise expressing in the cell a first exogenous genetic material, e.g., a first polynucleotide sequence comprising an immortalizing gene, the expression of which results in immortalization of the cell; in addition, one or more exogenous genetic material is expressed such that expression of the first polynucleotide is reduced or attenuated. In some embodiments, further expression of one or more exogenous genetic material (e.g., a second polynucleotide sequence) replaces the first exogenous material.
The method may comprise expressing the system in a cell. The system comprises an immortalizing gene. In some embodiments, the system comprises a first polynucleotide sequence comprising an immortalizing gene and a second polynucleotide sequence comprising a replacement gene to replace the immortalizing gene or a fragment thereof. In some embodiments, the system can be induced with a modulator to conditionally modulate immortalization of a cell.
In some embodiments, the method comprises expressing a system in the cell, the system comprising an immortalizing gene, and inducing the system with a modulator to conditionally modulate the immortalization of the cell. In some embodiments, the method comprises modulating immortalization of a cell by inducing at least one genetic modification in the cell.
In some embodiments, the methods comprise modulating immortalization of a cell by inducing an inactivating moiety that reduces expression of an immortalizing gene in the cell. Expression of the immortalizing gene can be reduced after induction of the inactivating moiety by the modulator. In some embodiments, the method comprises modulating immortalization of a cell by inducing a nucleic acid editing moiety that reduces expression of an immortalizing gene in the cell. Expression of the immortalizing gene can be reduced upon induction of the nucleic acid editing moiety by the modulator. In some embodiments, the methods comprise modulating immortalization of a cell by inducing a nucleic acid editing moiety that can exchange, replace, or delete an immortalizing gene in the cell. In some embodiments, the method comprises modulating immortalization of a cell by deletion or substitution of an immortalizing gene. In some embodiments, the method comprises expressing a first polynucleotide sequence comprising an immortalizing gene that induces cell immortalization.
In some embodiments, the method further comprises inducing a modulator inside the cell. In some embodiments, induction can be achieved by adding or administering an activator to the cell, wherein the activator (or activating moiety) can induce the modulator, thereby activating the system. The exchange between the immortalizing gene and the replacement gene is influenced by the induction system with the regulator.
In some embodiments, exchanging the immortalizing gene with a replacement gene reduces the expression of the immortalizing gene. The coding sequence of the immortalizing gene or fragment thereof and the coding sequence of the replacement gene may be flanked by one or more recombinase sites, and the modulator may be a recombinase that can affect the exchange between the immortalizing gene and the replacement gene, such that the immortalizing gene is completely or partially deleted, such that expression of the immortalizing gene is reduced or decreased upon modulator-mediated exchange of the immortalizing gene with the replacement gene. In some embodiments, the replacement gene may comprise a gene or fragment thereof that reduces the immunogenicity of the cell when introduced into a living organism and enhances the allogeneic or xenogeneic compatibility of the cell.
In one aspect, the present disclosure provides a method of genetically modifying a cell. The method comprises expressing the system inside a cell and inducing the immortalization of a regulatable cell. The system comprises a polynucleotide sequence comprising an immortalizing gene. In some embodiments, expression of the immortalizing gene is inactivated by an inactivating moiety. In some embodiments, the method comprises expressing an immortalizing gene and inducing an inactivating moiety in the cell to conditionally modulate immortalization of the cell. Expression of the immortalizing gene immortalizes the cell. In some embodiments, the system further comprises a nucleic acid editing moiety comprising a nucleic acid cleavage moiety. The nucleic acid editing moiety is capable of inducing at least one genetic modification in a cell. In some embodiments, the at least one genetic modification in the cell induces the inactivating moiety. In some embodiments, induction of the inactivating moiety inactivates expression of the immortalizing gene in the cell. In addition, the at least one genetic modification may enhance allogeneic or xenogeneic compatibility of the cell.
In one aspect, the present disclosure provides a method of making a cell comprising two or more different genetic modifications resulting from two or more genetic modification events following immortalization of the cell. The method includes introducing into a cell a polynucleotide sequence comprising an immortalizing gene, the expression of the immortalizing gene inducing immortalization of the cell, and performing two or more genetic modification events in the cell, wherein the two or more genetic modification events would otherwise shorten the lifespan of the cell in the absence of a system. In some embodiments, two or more genetic modification events of a cell in the absence of a system in the cell may be capable of shortening the lifespan of the cell. In some embodiments, the two or more genetic modification events comprise two or more sequential rounds of genetic modification. In some embodiments, at least one genetic modification of the two or more genetic modification events can be a germline modification. In one embodiment, one of the two or more genetic events can be a knock-in of a gene or portion thereof and one of the two or more genetic events can be a knock-out of a gene or portion thereof. In some embodiments, the first event affects a first gene, and the event is a gene knockout; and the second event is a tap-in. In some embodiments, the first gene encodes an incompatible allogeneic or xenogeneic epitope of the cell or a fragment thereof. In some embodiments, the second gene is a transgene, the translation product of which may be capable of enhancing allogenic or xenogenic compatibility of the cell. In some embodiments, the method further comprises inducing one or more effector moieties into the cell, wherein the one or more effector moieties can be a nuclease.
In some embodiments, the method comprises the step of immortalizing the cell by introducing an immortalizing gene into the cell. Exemplary immortalizing genes have been discussed in the preceding sections. In some cases, the immortalizing gene is introduced into the genome of the cell. In some cases, the immortalizing gene is introduced into a chromosome. In some cases, the immortalizing gene is introduced into a specific chromosomal locus, such as the AAVS1 locus in the genome of the cell. In some embodiments, the immortalizing gene may be integrated at the GGTA1 locus. In some embodiments, the immortalizing gene may be integrated at the CMAH locus. In some embodiments, the immortalizing gene may be integrated at the B4GALNT2 locus. In some embodiments, the immortalizing gene may be integrated at the B2M locus. In some embodiments, the immortalizing gene can be integrated at the ROSA26 locus. In some embodiments, the immortalizing gene may be integrated at the COLA1 locus. In some embodiments, the immortalizing gene can be integrated at the TIGRE locus.
One or more targeting vectors can be designed that comprise one or more polynucleotide sequences that are expressed in a cell. In some cases, the vector may comprise a polynucleotide sequence that may contain an immortalizing gene coding sequence, or a replacement gene coding sequence, or a polynucleotide sequence that encodes a modulator, or any combination thereof, wherein the modulator may be a recombinase. In some cases, the vector may comprise a polynucleotide sequence containing, for example, an immortalizing gene, such that the immortalizing gene is introduced into a particular desired locus of the genome. In addition, the one or more vectors may comprise one or more polynucleotide sequences operably linked to a promoter. The promoter may be an inducible promoter. In some cases, one or more polynucleotide sequences may comprise one or more termination sequences. Various such embodiments have been described elsewhere in this specification and may be suitably applied in this section.
In some embodiments, a plurality of vectors are produced, each comprising a polynucleotide sequence encoding an immortalizing gene, e.g., a first polynucleotide sequence, or a second polynucleotide sequence encoding a replacement gene or an inactivated gene, or a third polynucleotide sequence encoding a modulator, e.g., a recombinase gene. In some embodiments
In some embodiments, the first, second, and optionally third polynucleotide sequences (if present) are introduced into the cell by separate viral vectors. In some cases, one or more polynucleotide sequences are introduced by a non-viral vector.
In some embodiments, the second polynucleotide sequence may be introduced at a different time point than the introduction of the first polynucleotide sequence comprising the immortalizing gene. In some embodiments, the third polynucleotide sequence may be introduced at a different time point than the introduction of the second polynucleotide sequence. In some cases, the method comprises introducing a first vector at one time point, introducing a second vector at a second time point, and introducing a third vector at a third time point.
In some embodiments, the cell may be immortalized after expression of the immortalizing gene in the cell. In some embodiments, immortalization of a cell increases the number of cell cycles (or cell divisions) of the cell. In some embodiments, expression of the immortalizing gene increases the number of cell divisions by at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1000-fold, or more, as compared to the same cell that does not express the immortalizing gene.
In some embodiments, the method comprises introducing the second vector from 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 60 weeks, 70 weeks, 80 weeks, 90 weeks, or 100 weeks or more after the introduction of the vector comprising the immortalizing gene.
In some embodiments, the method comprises introducing the second vector after 2 cell cycles, 3 cell cycles, 4 cell cycles, 5 cell cycles, 10 cell cycles, 20 cell cycles, 30 cell cycles, 40 cell cycles, 50 cell cycles, 60 cell cycles, 70 cell cycles, 80 cell cycles, 90 cell cycles, 100 cell cycles, or more cell cycles.
In some embodiments, the first, second and third polynucleotide sequences are introduced into the genome of the cell. In some embodiments, the first, second, and third polynucleotide sequences are introduced into a chromosome of the cell. In some embodiments, the second and/or third polynucleotide sequences are not introduced into the chromosome of the cell.
In some embodiments, the first polynucleotide sequence and the second polynucleotide sequence, or corresponding vectors comprising them, are introduced into the cell simultaneously.
In some embodiments, the first polynucleotide sequence and the second polynucleotide sequence may be comprised in the same vector.
In some embodiments, the substitution or exchange of the second polynucleotide sequence is induced at a time interval after expression of the first polynucleotide sequence. In some embodiments, the substitution or exchange of the second polynucleotide sequence is induced by a modulator, an activator, or both. In some embodiments, the modulator is activated by an activator that is externally administered to a cell comprising the first and second polynucleotide sequences, which activates the modulator within the cell, and which in turn induces a substitution or exchange of the first polynucleotide sequence with the second polynucleotide sequence. In some embodiments, the modulator can induce expression of the second polynucleotide sequence.
In some embodiments, expression of the immortalizing gene is reduced upon introduction, exchange, or expression of the second polynucleotide sequence or the vector comprising the second polynucleotide sequence. In some cases, expression of the immortalizing gene is reduced by at least 2-fold as compared to expression of the immortalizing gene prior to inducing the second polynucleotide sequence. In some cases, expression is reduced by at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 16 fold, at least 17 fold, at least 18 fold, at least 19 fold, at least 20 fold, at least 21 fold, at least 22 fold, at least 23 fold, at least 24 fold, at least 25 fold, at least 26 fold, at least 27 fold, at least 28 fold, at least 29 fold, at least 30 fold, at least 31 fold, at least 32 fold, at least 33 fold, at least 34 fold, at least 35 fold, at least 36 fold, at least 37 fold, at least 38 fold, at least 39 fold, at least 40 fold, at least 41 fold, at least 42 fold, at least 43 fold, at least 44 fold, at least 45 fold, at least 46 fold, as compared to expression prior to exchange of the immortalizing gene for the second polynucleotide sequence, At least 47 times, at least 48 times, at least 49 times, or at least 50 times. In some cases, expression is reduced at least 55-fold, at least 60-fold, at least 65-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 85-fold, at least 90-fold, at least 95-fold, at least 100-fold, at least 105-fold, at least 110-fold, at least 115-fold, at least 120-fold, at least 125-fold, at least 130-fold, at least 135-fold, at least 140-fold, at least 145-fold, at least 150-fold, at least 155-fold, at least 160-fold, at least 165-fold, at least 170-fold, at least 175-fold, at least 180-fold, at least 185-fold, at least 190-fold, at least 200-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, as compared to expression prior to the exchange of the immortalizing gene with the second polynucleotide sequence, At least 47 times, at least 48 times, at least 49 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, or at least 100 times.
In some cases, expression of the immortalizing gene is reduced by up to 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, or up to 1000-fold compared to expression prior to the exchange of the second polynucleotide sequence.
In some embodiments, the reduction of the immortalizing gene may be from 1% to 100% of the level of expression prior to induction of the system, or from about 1% to 99%, or from about 5% to 90%, or from about 10% to 80%, or from about 20% to 70%, or from about 30% to 60%, or from about 40% to 50%, or from about 1% to 5%, 1% to 10%, 1% to 20%, 1% to 30%, 1% to 40%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, or from about 1% to 95% of the level of expression prior to induction of the system. In some cases, the reduction in immortalizing genes may be about 100% of the level of expression prior to induction of the system.
In one embodiment, two or more genetic modification events in a cell may undergo two or more sequential rounds of genetic modification. In some embodiments, two or more genetic modifications can be made over the course of at least 1 day, or over the course of at least 2 days, or at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, or more, or any time therebetween.
In one embodiment, the second genetic modification can be performed after the first genetic modification, wherein the interval between the two genetic modifications is at least 1 day or at least 2 days, or at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months or more, or any time in between.
In one aspect, the present disclosure provides a method wherein two or more different genetic modifications occur in the nucleus. In some embodiments, the method comprises preparing a genetically modified embryo. In some embodiments, the method comprises introducing a nucleus or derivative thereof into an embryo or embryonic precursor. In some embodiments, the embryo is an enucleated egg. In some embodiments, the egg may be fertilized prior to enucleation. In some embodiments, the egg may not be spermized prior to enucleation. In some embodiments, the egg cells may not be spermized prior to enucleation.
In some embodiments, the genetically modified cell is a blastocyst.
In one embodiment, provided herein are genetically modified cells. The genetically modified cell can be a genetically modified embryo. Also provided herein is a method of making a genetically modified embryo by expressing the system in a cell to induce at least one genetic modification in the nucleus.
In one aspect, the present disclosure provides a method for preparing a genetically modified embryo, the method comprising expressing a system in a cell to induce at least one modification in the nucleus. The cell may comprise a system. The system may comprise a polynucleotide sequence comprising an immortalizing gene, an inactivating moiety capable of inactivating expression of the immortalizing gene, a nucleic acid editing moiety comprising a cleavage moiety, wherein the nucleic acid editing moiety is capable of introducing at least one genetic modification in the nucleus. After at least one genetic modification in the nucleus has been performed, an inactivating moiety may be introduced which inactivates the expression of the immortalizing gene in the cell. The nucleus of the genetically modified cell may be introduced into an embryo or embryonic precursor. In some cases, the embryonic precursor is a single cell. In some cases, the embryonic precursors are dyads, tetrads, or 16 cell stages.
Alternatively, or in addition, genetically modified (engineered) cells, such as engineered egg cells, can be produced by performing one or more genetic modification events directly on a cell or embryo. In some embodiments, one or more genetic modifications can be performed by electroporation or by microinjection. In some embodiments, two or more genetic modifications may be made by any combination of these techniques.
In some aspects, the present disclosure provides a method for developing a transgenic animal comprising a transgene. Introducing a transgene into the cell, particularly into the nucleus, wherein the expressing transgene replaces an epitope that is incompatible with the transgenic animal and expresses an epitope that is compatible with the transgenic animal. In addition, the transgene may comprise a gene beneficial to the transgenic animal.
The transgenic animal can be a mammal, including a murine, porcine, bovine, canine, and/or feline mammal. In some embodiments, genetically modified animals can be prepared by methods or any combination of methods described anywhere in this specification. In some embodiments, the transgenic animal may comprise one or more genetic modifications described in the specification. In some embodiments, a transgenic animal can comprise one or more polynucleotide sequences described in the specification.
Cells
In one aspect, the present disclosure provides, inter alia, an isolated cell, tissue, organ or animal comprising a plurality of transgenes, wherein the plurality of transgenes comprises at least one inflammation response transgene or at least one immune modulator transgene. In some cases, the one or more transgenes comprise at least two or at least three transgenes selected from the group consisting of: an inflammatory response transgene, an immune response transgene, an immunomodulator transgene, or a combination thereof.
In one embodiment, the cell of the present disclosure is a mammalian cell.
In some embodiments, the cell may be a porcine cell.
In some embodiments, the cell may be a bovine cell.
In some embodiments, the cell may be a sheep cell.
In some embodiments, the cell can be an equine cell.
Alternatively, in some embodiments, the cell is a human cell.
In one aspect, the cell comprises a system as described in the preceding section. In some embodiments, the cell comprises one or more transgenes. In some embodiments, the cell comprises a first transgene expressed for a first period of time, after which expression of the first transgene is reduced or attenuated. In some embodiments, the expression of the first transgene is reduced or attenuated by expression of the second transgene. In some embodiments, the cell can express a third or fourth transgene. In some embodiments, the expression of one or more transgenes in a cell can be individually modulated. In some embodiments, the expression of each transgene in the cell can be individually regulated. In some embodiments, the expression of each transgene in a cell can be individually modulated by an activator that can be added to the cell. In some embodiments, a second or subsequent transgene may be expressed in the cell at a second, third, or subsequent time period, and then modulated. In some embodiments, the second or subsequent transgene may be permanently expressed.
In some embodiments, the first transgene may be comprised in a first polynucleotide sequence. In some embodiments, the second transgene may be comprised in a second polynucleotide sequence. In some embodiments, the third transgene may be comprised in a third polynucleotide sequence. In some embodiments, a polynucleotide sequence may comprise one or more transgenes.
In some embodiments, the first polynucleotide and the second polynucleotide are introduced into the cell by any vector described elsewhere in the disclosure. For example, in some cases, the first polynucleotide sequence can be introduced into the cell by infecting the cell with a viral vector, wherein the virus delivers a nucleic acid sequence into the cell, the nucleic acid comprising the first polynucleotide sequence. In some cases, the first polynucleotide sequence can be introduced into the genome of the cell. In some embodiments, the second polynucleotide sequence is delivered into the cell by a plasmid vector or a viral vector.
In some embodiments, the cell comprises a second or optional third polynucleotide sequence encoding, for example, a modulator that, upon induction with an activator, can affect the exchange of the first polynucleotide sequence with the second polynucleotide sequence.
In some embodiments, the second polynucleotide and the first polynucleotide are designed such that the first polynucleotide, flox, is within the coding sequence of the replacement gene for the second polynucleotide sequence. The immortalizing gene and the reporter gene (if present) can be expressed as long as the system does not activate Cre recombinase to act on the polynucleotide, thereby excising the loxP-loxP region comprising the first polynucleotide sequence and reannealing a second polynucleotide which can be subsequently expressed to replace the gene coding sequence.
Immortalization of a cell by expression of an immortalizing gene confers on the cell the ability to proliferate without altering other characteristics of the original primary cell. This is useful for increasing the number of cells of the desired terminally differentiated cell type, and the cells can be stable and can undergo one or more rounds of gene editing steps. In some cases, the immortalizing gene is introduced and expressed followed by the introduction of more than one gene, e.g., a second gene, a third gene, a fourth gene, or more genes.
In some cases, the cell undergoes multiple rounds of cell division after expression of the first polynucleotide sequence encoding the immortalizing gene. In some cases, the number of cells increases at least 2-fold, at least 4-fold, at least 6-fold, at least 8-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, or more after immortalization.
In some embodiments, the first time period comprises a time period comprising repeated cell cycles prior to introducing the second polynucleotide sequence into the immortalized cell. In some embodiments, the second polynucleotide sequence is introduced into the immortalized cell at least 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 22 days, 24 days, 26 days, 28 days, 30 days, more than 1 month, or more than 2 months, 3 months, 4 months, 5 months, or 6 months after the introduction of the first polynucleotide sequence.
In some embodiments, the cell may comprise a system such that expression of the second polynucleotide sequence may reduce or attenuate expression of the first polynucleotide sequence encoding the immortalizing gene. In some embodiments, the cell comprises a first polynucleotide sequence that is exchanged for a second polynucleotide sequence encoding, for example, a replacement gene by deletion of a sequence encoding a recombinase enzyme and insertion of a second polynucleotide sequence. In some cases, the exchange of the first polynucleotide sequence with the second polynucleotide sequence may be dependent on the induction and/or activation of the modulator by an activator.
In some embodiments, the cell may comprise a third polynucleotide sequence encoding a modulator or a second replacement gene. The third polynucleotide sequence may be introduced into the cell at the same time or after the introduction of the second polynucleotide sequence. The modulator may be an enzyme, such as Cre recombinase. In some cases, expression of the third polynucleotide sequence encoding the modulator may be dependent on administration of the activator from outside the cell. In some cases, the activator can bind to and activate the regulatory protein. In some cases, the activator is administered to the cell after delivery of the third polynucleotide and the cell is stabilized. In some cases, the activator is administered several hours after or within the same day as the introduction of the third nucleotide sequence. In some cases, the activator is administered about 1, 2, 3, 4, 5, 6, or 7 days or more after the introduction of the third polynucleotide sequence. In some cases, the activator is administered at any time when it is appropriate and/or desirable for the exchange to occur, thereby replacing the immortalizing gene with the replacement gene.
In some embodiments, the introduction and expression of the replacement gene reduces the expression of the immortalizing gene. In some cases, the introduction and expression of the replacement gene abolishes the expression of the immortalizing gene and restores the behavior of the cell to a non-immortalized phenotype.
In one aspect, the cells described herein are obtainable by the methods described in the present disclosure.
In some embodiments, a cell refers to a collection of cells having the same characteristics. In some embodiments, a cell refers to a collection of, e.g., at least 1X 10^6 cells, at least 1X 10^7 cells, at least 1X 10^8 cells, at least 1X 10^9 cells, at least 1X 10^10 cells, at least 1X 10^12 cells, which are allogeneic, and which comprise at least one exogenous polynucleotide as described above.
In some embodiments, the cell is a somatic cell. In some embodiments, the cell is a post-natal cell. In some embodiments, the cell is an adult cell (e.g., an adult ear fibroblast). In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a mammalian non-human cell. In some embodiments, the cell is a porcine cell.
In some embodiments, the cell is a human cell.
In some embodiments, the cell is a fetal/embryonic cell (e.g., an embryonic blastomere). In some embodiments, the cell is a germline cell. In some cases, the cell is an oocyte. In some embodiments, the cell is a stem cell. In some embodiments, the cell is a cell from a primary cell line. In some embodiments, the cell is selected from: epithelial cells, mesenchymal cells, hepatocytes, granulosa cells, adipocytes. In some embodiments, the cell is a fibroblast. In some embodiments, the fibroblast is a female fetal fibroblast. In some cases, the cell is in vivo.
In some embodiments, the cell is a single cell. In some embodiments, the cells are members of a cell colony.
In some embodiments, the cell is conditionally immortalized by expression of an immortalizing gene (e.g., a transgene encoding an immortalizing gene) and then restored to a non-immortalized state by modulation or inhibition of the transgene. In some cases, the modulation of the transgene is a conditional crossover or replacement transgene.
In some embodiments, the cell has modulated expression of an immortalizing transgene.
In some embodiments, the cell does not express an immortalized transgene.
In some embodiments, the cell expresses one or more transgenes that are not one or more immortalizing genes.
In some embodiments, the cell comprises a human gene. In some cases, the cell expresses a human gene.
In some embodiments, the cell expresses a gene that enhances the allogeneic or xenogeneic compatibility of the cell.
In some embodiments, the cell expresses one or more transgenes, each independently selected from the group consisting of: complement responsive transgenes (e.g., CD46, CD55, CD 59); a coagulation responsive transgene (e.g., CD39, THBD or TBM, TFPI); an inflammation response transgene (e.g., A20, HO-1, CD 47); an immune response transgene (e.g., HLA-E, B2M); and/or an immunomodulator transgene (e.g., PD-L1, FasL). In some cases, the cell expresses one or more human transgenes selected from the group consisting of: B2M, HLA-E, CD47, PDL1, CD46, CD55, CD59, A20, THBD, TFPI, CD39, EPCR, FASL, CIITA, CTLA-Ig and HO-1.
In some embodiments, the cell expresses one or more transgenes under the influence of an inducible promoter. In some cases, the cell expresses one or more transgenes under the influence of the ubiquitin promoter. In some cases, the cells express one or more transgenes flanked by loxP sites.
In one aspect, the compositions and methods described herein provide a safe and easy method for preparing genetically engineered cells from primary mammalian cells without the need for stem cell manipulation and/or dedifferentiation and differentiation procedures. In some cases, the compositions and methods described herein can be used to treat organ failure in humans using genetically modified porcine cells. For example, mammalian cells are conditionally immortalized using the methods described herein to obtain genetically modified cells suitable for use in vivo (e.g., in organ regeneration). In some cases, the mammalian cell is a non-human mammalian cell. For example, adult primary porcine cells are increased in number and appropriately genetically modified to reduce xenotoxicity and introduced into decellularized organ scaffolds for tissue formation. The organ may then be introduced into the recipient human.
Various other uses are contemplated using the compositions and methods described herein. In some cases, conditionally immortalized and appropriately genetically engineered cells can be prepared for cancer immunotherapy. The cells may be allogeneic or autologous delivery.
In another example, suitable cells can be generated using the methods and strategies described herein to treat inflammatory diseases. In some cases, the engineered cell obtained by the above methods may be a lymphocyte, such as a CD4+ or CD8+ T cell. The cell may express one or more cell surface proteins, such as a receptor, e.g., a TCR. In some cases, the inflammatory disease is an autoimmune disease. In some cases, as described in this paragraph, for example, the engineered cell may express one or more inhibitors or immunomodulators. In some cases, the cell can be an engineered Treg cell.
In another example, the compositions and methods described herein can be used to treat metabolic or genetic diseases, where corrective therapy can be initiated using an infected cell type and designed to express a corrective protein that is absent or deficient in the subject.
Reagent kit
Kits for making and using the systems are also provided. The kit may include at least one vector comprising a polynucleotide sequence comprising an immortalizing gene capable of being expressed in a mammalian cell; and an information delivery system including instructions for use of the contents and implementation of the method. In some cases, the kit may further comprise a polynucleic acid encoding a recombinase that recognizes a recombinase site of a vector comprising a polynucleotide sequence comprising an immortalizing gene. Kits may also comprise other components useful in the methods, for example, polynucleotide sequences encoding replacement genes, polynucleotide sequences encoding regulatory genes in one or more vectors; activators, reaction buffers, positive controls, negative controls, and the like. Suitable vectors may be lentiviral or adenoviral or adeno-associated vectors (AAV). The kit may further comprise cells comprising one or more polynucleic acids. The kit may also comprise a suitable medium for the cells. In some cases, the cells may be frozen cells. In some cases, the kit may include a vial containing cells frozen in liquid nitrogen. In some cases, a kit may comprise one or more components that require different temperature conditions for storage (as described in the specification).
One form in which the instructions may be present is information printed in the packaging of the kit, on a suitable medium or substrate such as a package insert, e.g., a sheet or sheets of paper on which the information is printed. Another approach is a computer readable medium, such as a floppy disk, CD, etc., on which information has been recorded. Another method that may exist is a web site that may be used via the internet to access information on a deleted site. Any convenient method may be present in the kit.
Examples
Example 1.Construction of exemplary polynucleotide sequences for modulating cell immortalization.
Targeting constructs for modulating cell immortalization can be designed and cloned into suitable vectors with multiple cloning sites. The targeting construct can be a polynucleic acid comprising the coding sequence of the TERT gene flanked by loxP sites (fig. 1A, 1B), and can be constructed as follows. The individual coding and non-coding sequences may be inserted or ligated in any order, as determined by one of skill in the art, and may depend, for example, on the selection and availability of restriction sites. The coding sequence of the TERT gene can be appropriately inserted into the clones, operably linked to a promoter suitable for the cell. In the given example, promoter mEF1a is linked upstream of TERT. The WPRE polyA termination sequence is ligated downstream of the coding sequence and upstream of the 3' Lox site. In addition, the reporter gene EGFP is inserted 5' to the TERT coding sequence and in frame so that both EGFP and TERT sequences are under the same promoter. The 2A peptide can be inserted between the EGFP and TERT coding sequences for post-translational cleavage of both proteins (reporter and TERT). The sequence can be inserted into any desired location of the genome as shown in FIG. 1B.
Claims (160)
1. A system for modulating cell immortalization comprising:
a first polynucleotide sequence comprising an immortalizing gene, wherein expression of said immortalizing gene results in the immortalization of said cell, and
a second polynucleotide sequence comprising a replacement gene replacing the immortalizing gene or a fragment thereof,
wherein expression of said immortalizing gene in said cell is reduced upon induction of said system with a modulator that affects the exchange between said immortalizing gene and said replacement gene.
2. The system of claim 1, wherein the first polynucleotide sequence is chromosomal.
3. The system of claim 2, wherein the first polynucleotide sequence is integrated at one or more sites in the genome of the cell, wherein the one or more sites are selected from the group consisting of: AAVS1 site, GGTA1 site, CMAH site, B4GALNT2 site, B2M site, ROSA26 site, COLA1 site and TIGRE site.
4. The system of claim 2, wherein the first polynucleotide sequence is integrated into the genome of the cell by a nucleic acid editing portion comprising a nucleic acid cleavage portion.
5. The system of any one of claims 1-4, wherein the replacement gene is capable of enhancing allogenic or xenogenic compatibility of the cells.
6. The system of claim 5, wherein the replacement gene encodes a human copy selected from the group consisting of: B2M, HLAE, CD47, PDL1, CD46, CD55, CD59, A20, THBD, TFPI, CD39, EPCR, FASL, CIITA, CTLA-Ig and HO-1.
7. The system of any one of claims 1-6, wherein the cells are incapable of expressing a functional copy of an incompatible allogeneic or xenogeneic epitope.
8. The system of claim 7, wherein the incompatible allogeneic or xenogeneic epitope is selected from the group consisting of: GGTA, CMAH, and B4 Gal.
9. The system of any one of claims 1-8, wherein the cell is a non-human mammalian cell.
10. The system of claim 9, wherein the cells are primary porcine cells.
11. The system of any one of claims 1-10, wherein the modulator is an enzyme.
12. The system of any one of claims 1-10, wherein (i) the immortalization gene in the first polynucleotide sequence is flanked by a first pair of recombinase sites, and (ii) the replacement gene in the second polynucleotide sequence is flanked by a second pair of recombinase sites, and wherein the modulator comprises a recombinase capable of causing the exchange.
13. The system of claim 12, wherein the first pair of recombinase sites and the second pair of recombinase sites comprise Lox sites, and wherein the recombinase is Cre recombinase.
14. The system of claim 12, wherein the first pair of recombinase sites and the second pair of recombinase sites comprise FRT sites, and wherein the recombinase is a Flp recombinase.
15. The system of claim 12, wherein the first pair of recombinase sites and the second pair of recombinase sites comprise attP and attB sites, and wherein the recombinase is a Φ C31 recombinase.
16. The system of any one of claims 1-15, further comprising a third polynucleotide sequence encoding the modulator, wherein the encoded modulator is capable of being activated upon induction of the system with an activator.
17. The system of claim 16, wherein the modulator is operably linked to an activator-inducible promoter of the third polynucleotide sequence.
18. The system of claim 16, wherein the modulator is part of a fusion protein comprising an activator-binding domain.
19. The system of claim 18, wherein the activator-binding domain is a ligand-binding domain of an estrogen receptor.
20. The system of any one of claims 16-19, wherein the activator comprises tamoxifen.
21. The system of any one of claims 1-20, wherein the immortalizing gene (i) is operably linked to a promoter, and (ii) flanks the promoter and an in-frame termination sequence.
22. The system of claim 21, wherein the first polynucleotide sequence further comprises a reporter sequence, wherein the reporter sequence (i) is operably linked to the promoter and (ii) flanks the promoter and the in-frame termination sequence.
23. The system of claim 22, wherein the immortalizing gene and the reporter sequence are linked by a protease cleavage sequence.
24. The system of any one of claims 1-23, further comprising a nucleic acid editing moiety comprising a nucleic acid cleavage moiety, wherein the nucleic acid editing moiety is capable of removing the immortalizing gene or fragment thereof from the first polynucleotide sequence to reduce the immortalizing gene in the cell.
25. The system of any one of claims 1-24, wherein the immortalizing gene is telomerase reverse transcriptase (TERT), SV 40T antigen, CDK4, HOXB, HOXA9, cMyc, Bmi1, or Myc T58A.
26. A cell comprising the system of any one of claims 1-25.
27. A kit comprising the system of any one of claims 1-25.
28. A method for modulating cell immortalization comprising:
(a) expressing the system in said cell; and
(b) inducing the system with a modulator to conditionally modulate the immortalization of the cell,
wherein the system comprises:
(i) a first polynucleotide sequence comprising said immortalizing gene, wherein expression of said immortalizing gene causes immortalization of said cell; and
(ii) a second polynucleotide sequence comprising a replacement gene replacing the immortalizing gene or fragment thereof,
wherein expression of said immortalizing gene in said cell is reduced upon induction of said system with a modulator that affects the exchange between said immortalizing gene and said replacement gene.
29. The method of claim 28, wherein the first polynucleotide sequence is chromosomal.
30. The method of claim 29, further comprising integrating the first polynucleotide sequence at one or more sites in the genome of the cell, wherein the one or more sites are selected from the group consisting of: AAVS1 site, GGTA1 site, CMAH site, B4GALNT2 site, B2M site, ROSA26 site, COLA1 site and TIGRE site.
31. The method of claim 29, further comprising integration of the first polynucleotide sequence into the genome of the cell by a nucleic acid editing moiety comprising a nucleic acid cleavage moiety.
32. The method of any one of claims 28-31, wherein the replacement gene is capable of enhancing allogenic or xenogenic compatibility of the cells.
33. The method of claim 32, wherein the replacement gene encodes a human copy selected from the group consisting of: B2M, HLAE, CD47, PDL1, CD46, CD55, CD59, A20, THBD, TFPI, CD39, EPCR, FASL, CIITA, CTLA-Ig and HO-1.
34. The method of any one of claims 28-33, wherein the cells are incapable of expressing a functional copy of an incompatible allogeneic or xenogeneic epitope.
35. The method of claim 34, further comprising modifying the incompatible allogeneic or xenogeneic epitope in the cell.
36. The method of claim 34, wherein the incompatible allogeneic or xenogeneic epitope is selected from the group consisting of: GGTA, CMAH, and B4 Gal.
37. The method of any one of claims 28-36, wherein the cell is a non-human mammalian cell.
38. The method of claim 37, wherein the cells are primary porcine cells.
39. The method of any one of claims 28-38, wherein the modulator is an enzyme.
40. The method of any one of claims 28-38, wherein (i) the immortalization gene in the first polynucleotide sequence is flanked by a first pair of recombinase sites, and (ii) the replacement gene in the second polynucleotide sequence is flanked by a second pair of recombinase sites, and wherein the modulator comprises a recombinase capable of causing the exchange.
41. The method of claim 40, wherein the first pair of recombinase sites and the second pair of recombinase sites comprise Lox sites, and wherein the recombinase is Cre recombinase.
42. The method of claim 40, wherein the first pair of recombinase sites and the second pair of recombinase sites comprise FRT sites, and wherein the recombinase is a Flp recombinase.
43. The method of claim 40, wherein the first pair of recombinase sites and the second pair of recombinase sites comprise attP and attB sites, and wherein the recombinase is a Φ C31 recombinase.
44. The method of any one of claims 28-43, wherein the system further comprises a third polynucleotide sequence encoding the modulator, the method further comprising inducing the cell with an activator to activate the encoded modulator.
45. The method of claim 44, wherein the modulator is operably linked to an activator-inducible promoter of the third polynucleotide sequence.
46. The method of claim 44, wherein the modulator is part of a fusion protein comprising an activator-binding domain.
47. A method according to claim 46, wherein the activator-binding domain is a ligand-binding domain of an estrogen receptor.
48. The method of any one of claims 44-47, wherein the activator comprises tamoxifen.
49. The method of any one of claims 28-48, wherein the immortalizing gene (i) is operably linked to a promoter and (ii) flanks the promoter and an in-frame termination sequence.
50. The method of claim 49, wherein the first polynucleotide sequence further comprises a reporter sequence, wherein the reporter sequence (i) is operably linked to the promoter and (ii) flanks the promoter and the in-frame termination sequence.
51. The method of claim 50, wherein the immortalizing gene and the reporter sequence are linked by a protease cleavage sequence.
52. The method of any one of claims 28-51, wherein the system further comprises a nucleic acid editing moiety comprising a nucleic acid cleavage moiety, wherein the nucleic acid editing moiety is capable of removing the immortalizing gene or fragment thereof from the first polynucleotide sequence to reduce the immortalizing gene in the cell.
53. The method of any one of claims 28-52, wherein the immortalizing gene is telomerase reverse transcriptase (TERT) or Myc T58A.
54. A system for genetically modifying a cell, comprising:
a polynucleotide sequence comprising an immortalizing gene, wherein expression of said immortalizing gene results in the immortalization of a cell and wherein said expression of said immortalizing gene is inactivated by an inactivating moiety, and
a nucleic acid editing portion comprising a nucleic acid cleavage portion, wherein said nucleic acid editing portion is capable of inducing at least one genetic modification in said cell, wherein said at least one genetic modification enhances allogenic or xenogenic compatibility of said cell,
wherein induction of said inactivating moiety inactivates expression of said immortalizing gene in said cell after said at least one genetic modification in said cell.
55. The system of claim 54, wherein the at least one genetic modification is a germline modification.
56. The system of claim 54, wherein the nucleic acid editing moiety is configured to complex with a target polynucleotide in the cell to induce the at least one genetic modification in the cell.
57. The system of claim 54, wherein the immortalizing gene in the polynucleotide sequence is flanked by a pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of deleting the immortalizing gene or fragment thereof from the polynucleotide sequence.
58. The system of claim 54, wherein the immortalizing gene in the polynucleotide sequence is in an active orientation and is flanked by a pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of inverting the immortalizing gene or fragment thereof into an inactive orientation.
59. A system according to claim 54, wherein (i) the immortalizing gene in the polynucleotide sequence is flanked by a pair of recombinase sites, and (ii) the system further comprises a further polynucleotide sequence comprising a replacement gene flanked by a further pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of causing an exchange between the immortalizing gene or fragment thereof and the replacement gene.
60. The system of claim 59, wherein the pair of recombinase sites and the additional pair of recombinase sites comprise Lox sites, and wherein the recombinase is Cre recombinase.
61. The system of claim 59, wherein the pair of recombinase sites and the additional pair of recombinase sites comprise FRT sites, and wherein the recombinase is a Flp recombinase.
62. A system according to claim 59, wherein said pair of recombinase sites and said further pair of recombinase sites comprise attP and attB sites, and wherein said recombinase is a Φ C31 recombinase.
63. A system according to claim 54, wherein (i) the polynucleotide sequence further comprises a pair of recombinase sites flanked by a promoter and the immortalizing gene, and (ii) the system further comprises a further polynucleotide sequence comprising a termination sequence flanked by a further pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of inserting the termination sequence between the pair of recombinase sites.
64. The system of any one of claims 57-63, further comprising a second polynucleotide sequence encoding the recombinase, wherein the encoded recombinase can be activated upon induction of the system with an activator.
65. The system of claim 64, wherein the recombinase is operably linked to an activator-inducible promoter of the second polynucleotide sequence.
66. The system of claim 64, wherein the recombinase is part of a fusion protein comprising an activator-binding domain.
67. A system according to claim 66, wherein the activator-binding domain is a ligand-binding domain of an estrogen receptor.
68. The system according to any one of claims 64-67, wherein the activator comprises tamoxifen.
69. The system of any one of claims 54-68, wherein the inactivating moiety comprises a further nucleic acid editing moiety comprising a further nucleic acid cleavage moiety, wherein the further nucleic acid editing moiety is capable of removing the immortalizing gene or fragment thereof from the polynucleotide sequence to inactivate expression of the immortalizing gene in the cell.
70. The system of any one of claims 54-69, wherein the polynucleotide sequence is chromosomal.
71. The system of claim 70, wherein the polynucleotide sequence is integrated at one or more sites in the genome of the cell, wherein the one or more sites are selected from the group consisting of: AAVS1 site, GGTA1 site, CMAH site, B4GALNT2 site, B2M site, ROSA26 site, COLA1 site and TIGRE site.
72. The system of claim 70, wherein the polynucleotide sequence is integrated in the genome of the cell by a nucleic acid editing portion comprising a nucleic acid cleavage portion.
73. The system of any one of claims 54-72, wherein the at least one genetic modification comprises deletion of a gene or fragment thereof encoding an incompatible allogeneic or xenogeneic epitope in the cell.
74. The system of claim 73, wherein the incompatible allogeneic or xenogeneic epitope is selected from the group consisting of: GGTA, CMAH, and B4 Gal.
75. The system of any one of claims 54-72, wherein the at least one genetic modification comprises addition of a gene encoding a human copy selected from the group consisting of: B2M, HLAE, CD47, PDL1, CD46, CD55, CD59, A20, THBD, TFPI, CD39, EPCR, FASL, CIITA, CTLA-Ig and HO-1.
76. The system of any one of claims 54-75, wherein the cell is a non-human mammalian cell.
77. The system of claim 76, wherein the cells are primary porcine cells.
78. The system of any one of claims 54-77, wherein the immortalizing gene is telomerase reverse transcriptase (TERT) or Myc T58A.
79. The system of any one of claims 54-78, wherein the at least one genetic modification comprises two or more different genetic modifications resulting from two or more genetic modification events.
80. The system of claim 79, wherein the two or more different genetic modifications comprise (i) knocking out a first gene encoding a first polypeptide, and (ii) knocking in a second gene encoding a second polypeptide.
81. The system of claim 80, wherein the second gene is a transgene.
82. A cell comprising the system of any one of claims 54-81.
83. A kit comprising the system of any one of claims 54-81.
84. A method of genetically modifying a cell, comprising:
(a) expressing a system in the cell to induce at least one genetic modification in the cell; and
(b) inducing an inactivating moiety in said cell to conditionally modulate immortalization of said cell,
wherein the system comprises:
(i) a polynucleotide sequence comprising an immortalizing gene, wherein expression of said immortalizing gene causes immortalization of said cell and wherein said expression of said immortalizing gene is inactivated by said inactivating moiety, and
(ii) a nucleic acid editing portion comprising a nucleic acid cleavage portion, wherein said nucleic acid editing portion is capable of inducing said at least one genetic modification in said cell, wherein said at least one genetic modification enhances allogenic or xenogenic compatibility of said cell,
wherein induction of said inactivating moiety inactivates expression of said immortalizing gene in said cell after said at least one genetic modification in said cell.
85. The method of claim 84, wherein the at least one genetic modification is a germline modification.
86. The method of claim 84, wherein the nucleic acid editing moiety is configured to complex with a target polynucleotide in the cell to induce the at least one genetic modification in the cell.
87. The method according to claim 84, wherein the immortalizing gene in the polynucleotide sequence is flanked by a pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of deleting the immortalizing gene or fragment thereof from the polynucleotide sequence.
88. The method of claim 84, wherein the immortalizing gene in the polynucleotide sequence is in an active orientation and is flanked by a pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of inverting the immortalizing gene or fragment thereof into an inactivating orientation.
89. A method according to claim 84 wherein (i) the immortalizing gene in the polynucleotide sequence is flanked by a pair of recombinase sites, and (ii) the system further comprises a further polynucleotide sequence comprising a replacement gene flanked by a further pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of causing an exchange between the immortalizing gene or fragment thereof and the replacement gene.
90. The method of claim 89, wherein the pair of recombinase sites and the additional pair of recombinase sites comprise Lox sites, and wherein the recombinase is Cre recombinase.
91. The method of claim 89, wherein the pair of recombinase sites and the additional pair of recombinase sites comprise FRT sites, and wherein the recombinase is a Flp recombinase.
92. A method according to claim 89, wherein said pair of recombinase sites and said additional pair of recombinase sites comprise attP and attB sites, and wherein said recombinase is a Φ C31 recombinase.
93. A method according to claim 84, wherein (i) the polynucleotide sequence further comprises a pair of recombinase sites flanking the promoter and the immortalizing gene, and (ii) the system further comprises a further polynucleotide sequence comprising a termination sequence flanked by a further pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of inserting the termination sequence between the pair of recombinase sites.
94. The method of any one of claims 87-93, wherein the system further comprises a second polynucleotide sequence encoding the recombinase, wherein the encoded recombinase can be activated upon induction of the system with an activator.
95. The method of claim 94, wherein the recombinase is operably linked to an activator-inducible promoter of the second polynucleotide sequence.
96. The method of claim 94, wherein the recombinase is part of a fusion protein comprising an activator-binding domain.
97. The method according to claim 96, wherein the activator-binding domain is a ligand-binding domain of an estrogen receptor.
98. The method according to any one of claims 94-97, wherein the activator comprises tamoxifen.
99. The method of any one of claims 84-98, wherein the inactivating moiety comprises a further nucleic acid editing moiety comprising a further nucleic acid cleavage moiety, wherein the further nucleic acid editing moiety is capable of removing the immortalizing gene or fragment thereof from the polynucleotide sequence to inactivate expression of the immortalizing gene in the cell.
100. The method of any one of claims 84-99, wherein the polynucleotide sequence is chromosomal.
101. The method of claim 100, further comprising integrating the polynucleotide sequence at one or more sites in the genome of the cell, wherein the one or more sites are selected from the group consisting of: AAVS1 site, GGTA1 site, CMAH site, B4GALNT2 site, B2M site, ROSA26 site, COLA1 site and TIGRE site.
102. The method of claim 100, further comprising integrating the polynucleotide sequence in the genome of the cell via a nucleic acid editing portion comprising a nucleic acid cleavage portion.
103. The method of any one of claims 84-102, wherein the at least one genetic modification comprises deletion of a gene encoding an incompatible allogeneic or xenogeneic epitope, or a fragment thereof, in the cell.
104. The method of claim 103, wherein said incompatible allogeneic or xenogeneic epitope is selected from the group consisting of: GGTA, CMAH, and B4 Gal.
105. The method of any one of claims 84-104, wherein the at least one genetic modification comprises addition of a human copy of a gene encoding a polypeptide selected from the group consisting of: B2M, HLAE, CD47, PDL1, CD46, CD55, CD59, A20, THBD, TFPI, CD39, EPCR, FASL, CIITA, CTLA-Ig and HO-1.
106. The method of any one of claims 84-105, wherein the cell is a non-human mammalian cell.
107. The method of claim 106, wherein the cells are primary porcine cells.
108. The method of any one of claims 84-107, wherein the immortalizing gene is telomerase reverse transcriptase (TERT) or Myc T58A.
109. The method of any one of claims 84-108, wherein the at least one genetic modification comprises two or more different genetic modifications resulting from two or more genetic modification events.
110. The method of claim 109, wherein the two or more different genetic modifications comprise (i) knocking out a first gene encoding a first polypeptide, and (ii) knocking in a second gene encoding a second polypeptide.
111. The method of claim 110, wherein the second gene is a transgene.
112. A method of making a cell comprising two or more different genetic modifications resulting from two or more genetic modification events, the method comprising:
(a) introducing into said cell a polynucleotide sequence comprising an immortalizing gene, wherein expression of said immortalizing gene results in immortalization of said cell; and
(b) performing two or more genetic modification events in the cell, wherein the two or more genetic modification events would otherwise shorten the lifespan of the cell in the absence of the system.
113. The method of claim 112, wherein the two or more genetic modification events comprise two or more sequential rounds of genetic modification.
114. The method of claim 114, wherein the at least one genetic modification is a germline modification.
115. The method of claim 112, wherein the two or more different genetic modifications comprise (i) knocking out a first gene and (ii) knocking in a second gene.
116. The method of claim 114, wherein the first gene encodes an incompatible allogeneic or xenogeneic epitope of the cell or a fragment thereof.
117. The method of claim 116, wherein the incompatible allogeneic or xenogeneic epitope is selected from the group consisting of: GGTA, CMAH, and B4 Gal.
118. The method of claim 114, wherein the second gene is a transgene.
119. The method of claim 114, wherein the second gene encodes a polypeptide capable of enhancing allo-or xeno-compatibility of the cell.
120. The method of claim 119, wherein the polypeptide is a human copy selected from the group consisting of: B2M, HLAE, CD47, PDL1, CD46, CD55, CD59, A20, THBD, TFPI, CD39, EPCR, FASL, CIITA, CTLA-Ig and HO-1.
121. The method of any one of claims 112-120, further comprising, in (b), inducing one or more actuator moieties into the cell, each of the one or more actuator moieties comprising a nuclease, wherein the one or more actuator moieties are capable of performing the two or more genetic modification events.
122. The method of any one of claims 112-121, wherein the expression of the immortalizing gene is inactivated by an inactivating moiety, and the method further comprises, after (b), inducing the inactivating moiety into the cell to inactivate the expression of the immortalizing gene in the cell.
123. The method according to claim 122, wherein the immortalizing gene in the polynucleotide sequence is flanked by a pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of deleting the immortalizing gene or fragment thereof from the polynucleotide sequence.
124. The method of claim 122, wherein the immortalizing gene in the polynucleotide sequence is in an active orientation and is flanked by a pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of inverting the immortalizing gene or fragment thereof into an inactivating orientation.
125. A method according to claim 122, wherein (i) the immortalizing gene in the polynucleotide sequence is flanked by a pair of recombinase sites, and (ii) the system further comprises a further polynucleotide sequence comprising a replacement gene flanked by a further pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of causing an exchange between the immortalizing gene or fragment thereof and the replacement gene.
126. The method of claim 125, wherein the pair of recombinase sites and the additional pair of recombinase sites comprise Lox sites, and wherein the recombinase is Cre recombinase.
127. The method of claim 125, wherein the pair of recombinase sites and the additional pair of recombinase sites comprise FRT sites, and wherein the recombinase is a Flp recombinase.
128. A method according to claim 125, wherein said pair of recombinase sites and said additional pair of recombinase sites comprise attP and attB sites, and wherein said recombinase is a Φ C31 recombinase.
129. A method according to claim 122, wherein (i) the polynucleotide sequence further comprises a pair of recombinase sites flanking the promoter and the immortalizing gene, and (ii) the system further comprises a further polynucleotide sequence comprising a termination sequence flanked by a further pair of recombinase sites, and wherein the inactivating moiety comprises a recombinase capable of inserting the termination sequence between the pair of recombinase sites.
130. The method of any one of claims 123-129, wherein the system further comprises a second polynucleotide sequence encoding the recombinase, wherein the encoded recombinase can be activated upon induction of the system with the activator.
131. The method of claim 130, wherein the recombinase is operably linked to an activator-inducible promoter of the second polynucleotide sequence.
132. The method of claim 130, wherein the recombinase is part of a fusion protein comprising an activator-binding domain.
133. The method according to claim 132, wherein the activator-binding domain is a ligand-binding domain of an estrogen receptor.
134. The method of any one of claims 130-133, wherein the activator comprises tamoxifen.
135. The method of any one of claims 122-134, wherein the inactivating moiety comprises a further nucleic acid editing moiety comprising a further nucleic acid cleavage moiety, wherein the further nucleic acid editing moiety is capable of removing the immortalizing gene or fragment thereof from the polynucleotide sequence to inactivate expression of the immortalizing gene in the cell.
136. The method of any one of claims 112-135, wherein the cell is a non-human mammalian cell.
137. The method of claim 136, wherein the cells are primary porcine cells.
138. The method of any one of claims 112-137, wherein the immortalizing gene is telomerase reverse transcriptase (TERT) or Myc T58A.
139. The method of any one of claims 112-138, wherein the two or more different genetic modifications occur in the nucleus of the cell, the method further comprising:
preparing a genetically modified embryo by introducing said nucleus of said cell or a derivative thereof into an embryo or an embryo precursor.
140. The method of claim 139, wherein said embryonic precursor is an enucleated egg, wherein said introducing said nucleus into said enucleated egg produces said genetically modified embryo.
141. The method of claim 139, wherein the embryo is a blastocyst, and wherein the preparation of the genetically modified embryo is performed by introducing the cell or derivative thereof into the inner cell mass of the blastocyst.
142. The method of any one of claims 139-140, further comprising developing the genetically modified embryo into a transgenic animal comprising the at least one genetic modification.
143. A genetically modified embryo made by the method of any one of claims 139-142.
144. A method of making a genetically modified embryo comprising:
(a) expressing in a cell a system to induce at least one genetic modification in the nucleus of said cell, wherein said system comprises:
(i) a polynucleotide sequence comprising an immortalizing gene, wherein expression of said immortalizing gene causes immortalization of said cell and wherein said expression of said immortalizing gene is inactivated by an inactivating moiety, and
(ii) a nucleic acid editing moiety comprising a nucleic acid cleavage moiety, wherein the nucleic acid editing moiety is capable of inducing the at least one genetic modification in the nucleus of the cell,
wherein introduction of said inactivating moiety inactivates expression of said immortalizing gene in said cell following said at least one genetic modification in said cell;
(b) introducing the inactivating moiety to inactivate immortalization of the cell; and
(c) preparing said genetically modified embryo by introducing said nucleus of said cell or a derivative thereof into an embryo or an embryo precursor.
145. The method of claim 144, wherein said embryonic precursor is an enucleated egg, wherein introduction of said nucleus into said enucleated egg results in preparation of said genetically modified embryo.
146. The method of claim 144, wherein the embryo is a blastocyst, wherein the preparation of the genetically modified embryo is performed by introducing the cell or derivative thereof into the inner cell mass of the blastocyst.
147. The method of any one of claims 144-146, further comprising developing the genetically modified embryo into a transgenic animal comprising the at least one genetic modification.
148. The method of any one of claims 144-147, wherein the at least one genetic modification is a germline modification.
149. The method of any one of claims 144-148, wherein the inactivating moiety is a recombinase capable of inducing the inactivation of the immortalizing gene by: (i) a deletion of the immortalizing gene or fragment thereof, (ii) an inversion of the immortalizing gene or fragment thereof, (iii) an exchange of the immortalizing gene or fragment thereof with a further polynucleotide sequence, or (iv) an insertion of a termination sequence upstream of the immortalizing gene.
150. The method of any one of claims 144-148, wherein the inactivating moiety is a further nucleic acid editing moiety comprising a further nucleic acid cleavage moiety, wherein the further nucleic acid editing moiety is capable of removing the immortalizing gene or fragment thereof from the polynucleotide sequence.
151. The method of any one of claims 144-150, wherein the polynucleotide sequence is chromosomal.
152. The method of claim 151, further comprising integrating the polynucleotide sequence at one or more sites in the genome of the cell, wherein the one or more sites are selected from the group consisting of: AAVS1 site, GGTA1 site, CMAH site, B4GALNT2 site, B2M site, ROSA26 site, COLA1 site and TIGRE site.
153. The method of claim 151, further comprising integrating the polynucleotide sequence in the genome of the cell via a nucleic acid editing portion comprising a nucleic acid cleavage portion.
154. The method of any one of claims 144-153, wherein the at least one genetic modification comprises deletion of a gene encoding an incompatible allogeneic or xenogeneic epitope or fragment thereof in the cell.
155. The method of claim 154, wherein the incompatible allogeneic or xenogeneic epitope is selected from the group consisting of: GGTA, CMAH, and B4 Gal.
156. The method of any one of claims 144-153, wherein the at least one genetic modification comprises addition of a gene encoding a human copy selected from the group consisting of: B2M, HLAE, CD47, PDL1, CD46, CD55, CD59, A20, THBD, TFPI, CD39, EPCR, FASL, CIITA, CTLA-Ig and HO-1.
157. The method of any one of claims 144-156, wherein the cell is a non-human mammalian cell.
158. The method of claim 157, wherein the cells are primary porcine cells.
159. The method of any one of claims 144-158, wherein the immortalizing gene is telomerase reverse transcriptase (TERT) or Myc T58A.
160. A genetically modified embryo made by the method of any one of claims 144-159.
Applications Claiming Priority (12)
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CNPCT/CN2020/077533 | 2020-03-03 | ||
CN2020077533 | 2020-03-03 | ||
CNPCT/CN2020/077557 | 2020-03-03 | ||
CN2020077535 | 2020-03-03 | ||
CNPCT/CN2020/077545 | 2020-03-03 | ||
CN2020077536 | 2020-03-03 | ||
CN2020077547 | 2020-03-03 | ||
CNPCT/CN2020/077535 | 2020-03-03 | ||
CN2020077557 | 2020-03-03 | ||
CN2020077545 | 2020-03-03 | ||
CNPCT/CN2020/077536 | 2020-03-03 | ||
CNPCT/CN2020/077547 | 2020-03-03 |
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CN114231480A (en) * | 2021-12-15 | 2022-03-25 | 复旦大学附属中山医院 | Human lung specific channel cell line |
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