Patent Application
20250236890
This application claims priority to Chinese Patent Application No. 202310380978.4, filed with the China National Intellectual Property Administration on Apr. 11, 2023, the entire contents of which are hereby incorporated by reference in their entirety for all purpose.
The present application relates to the field of molecular biology, and specifically to a non-LTR retrotransposon system and use thereof. The present application further specifically relates to: an isolated functional protein encoded by a retrotransposon, a nucleic acid encoding the functional protein, a nucleic acid set and a nucleic acid construct, and a composition, a recombinant vector, a recombinant host cell and a kit comprising the functional protein. The present application further specifically relates to: a method for introducing an exogenous nucleic acid fragment into the genome of a host cell, a method for editing the genome of a host cell, and a method for obtaining a host cell containing an exogenous nucleic acid fragment in the genome. The present application further specifically relates to use of the functional protein, the nucleic acid, the nucleic acid set, the nucleic acid construct, the composition, the recombinant vector, or the recombinant host cell for introducing an exogenous nucleic acid fragment gene into the genome of a host cell or preparing a drug or a preparation for gene therapy, cell therapy, genome research, or stem cell induction and post-induction differentiation.
A transposon is a DNA sequence that can be inserted or removed within the genome to transfer its own sequence or a complete copy of its own sequence within or between genomes (Mobile DNA III). Transposons are mainly divided into two categories. Among them, type I retrotransposons use RNA as an intermediate. In the process of moving from a donor site to a new insertion site, type I retrotransposons require the host's RNA polymerase to transcribe the transposable element into RNA, and then the reverse transcriptase domain encoded by the retrotransposon reverse-transcribes the RNA copy of the transposable element into DNA which is inserted into a new site. During the retrotransposition process of this type of retrotransposons, the sequence of the donor site would not be cleaved from the original site, so a plurality of copies of the retrotransposable element may be present in the genome.
According to different replication and integration mechanisms, retrotransposons can be divided into two categories: LTR (Long Terminal Repeat) and non-LTR. The latter is a type of mobile genetic element widely distributed in eukaryotic cell genomes. According to the characteristics of the nuclease functional domain contained, non-LTR retrotransposons can be further divided into two categories: apurinic/apyrimidinic nuclease (APE-type) and restriction enzyme-like nuclease (RLE-type). Among them, R2 family retrotransposons are a major representative of the RLE type non-LTR retrotransposons. Some R2 retrotransposons have high target site specificity and would specifically integrate into the gene encoding 28s rRNA.
Gene insertion and integration of large fragments have important application value in fields such as gene therapy, molecular breeding of animals and plants, and engineering of industrial microorganisms. Currently, there is a lack of effective tools and systems for insertion and integration of a large fragment gene in the industry. In recent years, the scientific community has developed some tools and methods capable of inserting and integrating a large fragment gene, but these methods still have some problems. For example, in cellular immunotherapy and gene therapy for hereditary diseases, lentivirus or retrovirus are most commonly used to integrate gene sequences, and based on this, there are several therapeutic products for the treatment of tumors and genetic disorders (Aiuti, A., Roncarolo, M. G. and Naldini, L. (2017) Gene therapy for ADA-SCID, the first marketing approval of an ex vivo gene therapy in Europe: paving the road for the next generation of advanced therapy medicinal products. EMBO Mol. Med. 9, 737 740; Aiuti, A. et al. (2009) Gene therapy for immunodeficiency due to adenosine deaminase deficiency. N. Engl. J. Med. 360, 447 458). However, using viruses to integrate a large fragment gene has some potential application limitations: first, the randomness of virus integration in the genome creates the risk of cancer; second, the size of an exogenous gene the virus can carry is also limited, which is not conducive to the transfer of a therapeutic large fragment gene; third, the immunogenicity of the virus may affect the long-term expression of an exogenous therapeutic gene and re-administration; fourth, the production of viruses needs the help of living cells, which makes the quality control and downstream processing of such products more complicated and more expensive, and has certain disadvantages in terms of industrialization. Therefore, non-viral large fragment integration can avoid various disadvantages caused by viral integration and become a valuable tool in gene therapy.
As a non-viral gene integration tool, retrotransposons can achieve the integration in the host genome and stable expression of a large fragment of an exogenous gene through RNA delivery, which can not only reduce negative effects such as immunogenicity, but can also be combined with LNP and other delivery technologies to directly treat target cells in vivo. Due to the lack of relevant retrotransposon tools, there are few applications of retrotransposon technology in gene therapy or other fields. There are few companies in the world that are mining a large number of active retrotransposons and trying to develop them into therapeutic tools, therefore, it is necessary to quickly discover and obtain more active retrotransposon products, and verify and detect their functions to provide more options for the development of gene therapy strategies.
It should be noted that methods described in this section are not necessarily methods that have been previously conceived or employed. It should not be assumed that any of the methods described in this section is considered to be the prior art just because they are included in this section, unless otherwise indicated expressly. Similarly, the problem mentioned in this section should not be considered to be universally recognized in any prior art, unless otherwise indicated expressly.
Based on this, in order to seek more advanced and more effective non-viral gene integration tools, the present application provides a non-LTR retrotransposon system and use thereof. The present application further specifically provides an isolated functional protein encoded by a retrotransposon, wherein, the functional protein has a functional protein sequence selected from the following (i) or a variant sequence of the aforementioned functional protein with functional protein activity in (ii)-(iv): (i) at least one amino acid sequence as shown in any one of SEQ ID NO: 1-94; (ii) at least one sequence having deletion, substitution, insertion, mutation of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the amino acid sequence as shown in any one of SEQ ID NO: 1-94; (iii) at least one amino acid sequence having at least 70%, 80%, 90%, 95% or 99% identity with the amino acid sequence as shown in any one of SEQ ID NO: 1-94; and (iv) at least one sequence obtained by further fusing the amino acid sequence as shown in any one of SEQ ID NO: 1-94 with other sequences. The functional protein encoded by the retrotransposon provided in the present application has high retrotransposition activity and can provide more options for the development of gene integration tools.
According to an embodiment of the present application, an isolated functional protein encoded by a retrotransposon can be provided, wherein, the functional protein includes the amino acid sequence as shown in formula (I):
C(X1)aC(X2)bH(X3)cH (I).
According to an embodiment of the present application, an isolated functional protein encoded by a retrotransposon can be provided, wherein, the functional protein includes the amino acid sequence as shown in formula (II):
G(X4)dQGD(X5)eS(X6)fF(X7)gD (II),
According to an embodiment of the present application, an isolated functional protein can be provided, wherein, the functional protein includes the amino acid sequence as shown in formula (III):
C(X8)hC(X9)iE(X10)jH(X11)kC(X12)lRH(X13)mPD(X14)n(X15)(X16)oK(X17)pY (III),
According to an embodiment of the present application, an isolated functional protein encoded by a retrotransposon is provided, wherein, the functional protein includes at least two of the amino acid sequences as shown in formula (I), formula (II), and formula (III).
According to an embodiment of the present application, an isolated functional protein encoded by a retrotransposon is provided, wherein, the functional protein includes the amino acid sequences as shown in formula (I), formula (II), and formula (III).
According to an embodiment of the present application, a nucleic acid can be provided, wherein, the nucleic acid encodes the functional protein described in the present application.
According to an embodiment of the present application, a nucleic acid set can be provided, the nucleic acid set includes a 5′-untranslated region, wherein, the 5′-untranslated region includes at least one of the nucleotide sequences as shown in SEQ ID NO: 95-188.
According to an embodiment of the present application, a nucleic acid set can be provided, the nucleic acid set includes a 3′-untranslated region, wherein, the 3′-untranslated region includes at least one of the nucleotide sequences as shown in SEQ ID NO: 189-282.
According to an embodiment of the present application, a nucleic acid set can be provided, the nucleic acid set includes a 5′-untranslated region and a 3′-untranslated region, wherein, the 5′-untranslated region includes the nucleotide sequence as shown in any one of SEQ ID NO: 95-188 or a variant thereof, and the 3′-untranslated region includes the nucleotide sequence as shown in any one of SEQ ID NO: 189-282 or a variant thereof, the RNA transcribed from the nucleic acid set can bind to the functional protein encoded by a specific retrotransposon.
According to an embodiment of the present application, a nucleic acid construct can be provided, the nucleic acid construct includes the nucleic acid described in the present application and/or the nucleic acid set described in the present application.
According to an embodiment of the present application, a composition may be provided, wherein, the composition includes: a functional protein or a functional fragment thereof encoded by a R2 family retrotransposon, or a nucleic acid encoding the functional protein or the functional fragment thereof, the functional protein or the functional fragment thereof has the function of catalyzing the insertion of an exogenous nucleic acid fragment into the genome of a cell; and a nucleic acid set, the nucleic acid set can be recognized by a functional protein or a functional fragment thereof encoded by a specific retrotransposon.
According to an embodiment of the present application, a recombinant vector can be provided, wherein, the recombinant vector includes the nucleic acid encoding the functional protein described in the present application, the nucleic acid described in the present application, and the nucleic acid set described in the present application, the nucleic acid construct described in the present application, or the composition described in the present application.
According to an embodiment of the present application, a recombinant host cell can be provided, wherein, the recombinant host cell comprises the functional protein described in the present application, the nucleic acid encoding the functional protein described in the present application, the nucleic acid described in the present application, and the nucleic acid set described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, or the recombinant vector described in the present application.
According to an embodiment of the present application, a method for introducing an exogenous nucleic acid fragment into the genome of a host cell can be provided, wherein, the method comprises: delivering the functional protein described in the present application, the nucleic acid encoding the functional protein described in the present application, the nucleic acid described in the present application, and the nucleic acid set described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, or the recombinant vector described in the present application into the host cell.
According to an embodiment of the present application, a method for editing the genome of a host cell can be provided, wherein, the method comprises: delivering the functional protein described in the present application, the nucleic acid encoding the functional protein described in the present application, the nucleic acid described in the present application, and the nucleic acid set described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, or the recombinant vector described in the present application into the host cell.
According to an embodiment of the present application, a method for obtaining a host cell containing an exogenous nucleic acid fragment in the genome can be provided, wherein, the method comprises: delivering the functional protein described in the present application, the nucleic acid encoding the functional protein described in the present application, the nucleic acid described in the present application, the nucleic acid set described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, or the recombinant vector described in the present application into the host cell.
According to an embodiment of the present application, use of the functional protein described in the present application, the nucleic acid encoding the functional protein described in the present application, the nucleic acid described in the present application, the nucleic acid construct described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, the recombinant vector described in the present application, or the recombinant host cell described in the present application for introducing an exogenous nucleic acid fragment into the genome of a host cell can be provided.
According to an embodiment of the present application, use of the functional protein described in the present application, the nucleic acid encoding the functional protein described in the present application, the nucleic acid described in the present application, the nucleic acid set described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, the recombinant vector described in the present application, or the recombinant host cell described in the present application for gene therapy, cell therapy, genomic research, or stem cell induction and post-induction differentiation can be provided.
According to an embodiment of the present application, a kit can be provided, wherein, the kit includes the functional protein described in the present application, the nucleic acid encoding the functional protein described in the present application, the nucleic acid described in the present application, the nucleic acid set described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, the recombinant vector described in the present application, or the recombinant host cell described in the present application.
It should be understood that the content described in this section is not intended to identify critical or important features of the examples of the present application, and is not used to limit the scope of the present application. Other features of the present application will be easily understood through the following description.
The accompanying drawings exemplarily show embodiments and form a part of the specification, and are used to explain exemplary implementations of the embodiments together with a written description of the specification. The embodiments shown are merely for illustrative purposes and do not limit the scope of the claims. Throughout the accompanying drawings, the same reference numerals denote similar but not necessarily same elements.
Unless otherwise indicated or contradicts the context, the terms or expressions used herein should be read in conjunction with the entire content of the present disclosure and as understood by those of ordinary skill in the art. All technical and scientific terms used herein have the same meanings as commonly understood by those of ordinary skill in the art, unless otherwise defined.
In the present application, the terms “nucleic acid” and “polynucleotide” are used interchangeably, and refer to polymerization forms of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogs thereof.
In the present application, the terms “polypeptide” and “peptide” are used interchangeably, and refer to polymers of amino acids of any length. Therefore, polypeptides, oligopeptides, proteins, antibodies and enzymes are all included in the definition of polypeptide.
As described in the present application, the “fragment” of a sequence refers to a portion of a sequence. For example, the fragment of a nucleic acid sequence refers to a portion of the nucleic acid sequence, and the fragment of an amino acid sequence refers to a portion of the amino acid sequence.
As described in the present application, a “variant” of a sequence is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleic acid sequence from another reference polynucleotide, and the differences in nucleic acid sequence may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, the differences are limited so that the sequences of the reference polypeptide and the variant are generally very similar, and are identical in many regions. A variant polypeptide and a reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. The substituted or inserted amino acid residue may or may not be a residue encoded by the genetic code. Variants of polynucleotides or polypeptides may be naturally occurring, such as allelic variations, or they may be unknown naturally occurring variants. Non-naturally occurring polynucleotide and polypeptide variants can be produced by mutagenesis techniques, direct synthesis, and other recombinant methods known to the skilled artisan.
The expression “functional protein encoded by a retrotransposon” as used in the present application refers to a polypeptide that catalyzes the integration of an exogenous nucleic acid fragment into a target site (such as genome or extrachromosomal DNA).
The term “exogenous nucleic acid fragment” used in the present application includes any gene of interest or any gene or fragment thereof that is transposable. In some non-limiting embodiments, the exogenous nucleic acid fragment is of a different origin than the host cell, for example, a nucleic acid sequence isolated from an organism different from the host cell, i.e., the exogenous nucleic acid fragment is exogenous to the host cell.
The terms “domain” and “functional domain” as used in the present application are used interchangeably and refer to the structure of a biomolecule that contributes to a specific function of the biomolecule, and may include a contiguous region of the biomolecule (such as a contiguous sequence) or different non-contiguous regions (e.g., non-contiguous sequences). Examples of protein functional domains include, but are not limited to, DNA binding domains, RNA binding domains, reverse transcriptase functional domains, and nuclease functional domains.
The term “Untranslated region” as used in the present application refer to a nucleic acid sequence located at both ends of a transposable element and flanking a transposable nucleic acid sequence. Among them, the untranslated region located at the 5′ end of the transposable nucleic acid sequence is called the 5′-untranslated region, and the untranslated region located at the 3′ end of the transposable nucleic acid sequence is called the 3′-untranslated region. In some embodiments, the RNA transcribed from the untranslated region can bind to the functional protein encoded by a specific retrotransposon.
The term “nucleic acid construct” as used in the present application is defined as a single-stranded or double-stranded nucleic acid molecule herein, and preferably refers to an artificially constructed nucleic acid molecule. Optionally, the nucleic acid construct further includes one or more operably linked regulatory sequences, which can direct the expression of a coding sequence in a suitable host cell under compatible conditions. The term “expression” is understood to include any step involved in the production of a protein or polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification and secretion. The term “regulatory sequence” includes all components necessary or advantageous for expression of the polypeptide/protein of the present application. Each regulatory sequence may be naturally present or exogenous to the nucleic acid sequence encoding the protein or polypeptide. These regulatory sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences should include promoters and initiation and termination signals for transcription and translation. Regulatory sequences with linkers can be provided for the purpose of introduction into specific restriction sites for linking the regulatory sequences to the coding region of a nucleic acid sequence encoding a protein or polypeptide.
The term “promoter” as used in the present application refers to a polynucleotide sequence that can control the transcription of a coding sequence. Promoter sequences include specific sequences sufficient to enable RNA polymerase to recognize, bind, and initiate transcription. In addition, promoter sequences may include sequences that optionally modulate the recognition, binding and transcription initiation activities of RNA polymerase in the nucleic acid construct provided in the present application. A promoter can affect the transcription of a gene located on the same nucleic acid molecule as the promoter or a gene located on a different nucleic acid molecule from the promoter.
The term “host cell” as used in the present application include, but are not limited to, an animal cell, a plant cell, an algal cell, a fungal cell, a yeast cell, or a bacterial cell. This term includes a progeny of an original cell into which an exogenous nucleic acid fragment has been introduced. Exemplary host cell includes human embryonic kidney cell HEK293T. It is understood that, due to natural, accidental or intentional mutations, the progeny of a single parent cell may not necessarily be identical to the original parent morphologically or in terms of genome or total DNA complement.
The term “vector” as used in the present application refers to a nucleic acid molecule capable of transporting another nucleic acid molecule connected to it. Examples of vectors include, but are not limited to, plasmids, viruses, bacteria, phages, and insertable DNA fragments. The term “plasmid” refers to a circular double-stranded DNA capable of accepting an exogenous nucleic acid fragment and replicating in prokaryotic or eukaryotic cells.
The present application provides a non-LTR retrotransposon system and use thereof. According to an embodiment of the present application, an isolated functional protein encoded by a retrotransposon can be provided, wherein, the functional protein has a functional protein sequence selected from the following (i) or a variant sequence of the aforementioned functional protein with functional protein activity in (ii)-(iv): (i) at least one amino acid sequence as shown in any one of SEQ ID NO: 1-94; (ii) at least one sequence having deletion, substitution, insertion, mutation of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the amino acid sequence as shown in any one of SEQ ID NO: 1-94; (iii) at least one amino acid sequence having at least 70%, 80%, 90%, 95% or 99% identity with the amino acid sequence as shown in any one of SEQ ID NO: 1-94; and (iv) at least one sequence obtained by further fusing the amino acid sequence as shown in any one of SEQ ID NO: 1-94 with other sequences.
According to an embodiment of the present application, an isolated functional protein encoded by a retrotransposon can be provided, wherein, the functional protein includes the amino acid sequence as shown in formula (I):
C(X1)aC(X2)bH(X3)cH (I).
According to an embodiment of the present application, an isolated functional protein encoded by a retrotransposon can be provided, wherein, the functional protein includes the amino acid sequence as shown in formula (II):
G(X4)dQGD(X5)eS(X6)fF(X7)gD (II),
According to an embodiment of the present application, an isolated functional protein can be provided, wherein, the functional protein includes the amino acid sequence as shown in formula (III):
C(X8)hC(X9)iE(X10)jH(X11)kC(X12)lRH(X13)mPD(X14)n(X15)(X16)oK(X17)pY (III),
According to an embodiment of the present application, an isolated functional protein encoded by a retrotransposon is provided, wherein, the functional protein includes at least two of the amino acid sequences as shown in formula (I), formula (II), and formula (III).
According to an embodiment of the present application, an isolated functional protein encoded by a retrotransposon is provided, wherein, the functional protein includes the amino acid sequences as shown in formula (I), formula (II), and formula (III).
In some embodiments, the retrotransposon is a non-LTR (Non-Long Terminal Repeat) retrotransposon. In some embodiments, the non-LTR retrotransposon is a restriction enzyme-like nuclease (RLE) retrotransposon. In some embodiments, the retrotransposon is a R2 family retrotransposon.
In some embodiments, the functional protein includes a DNA binding domain that can bind to a specific region in the genome of a cell, preferably, the DNA binding domain can bind to a gene encoding 28s rRNA. In some non-limiting examples, the functional protein takes the 28s rRNA gene as a target site. In some non-limiting examples, the functional protein takes the non-28s rRNA gene as a target site.
In some embodiments, the functional protein further includes an RNA binding domain, a reverse transcriptase functional domain, and/or a nuclease functional domain. In some non-limiting examples, the RNA binding domain of the functional protein associates with the RNA transcribed by the retrotransposon to form RNP (Ribonucleoprotein). In some non-limiting embodiments, the functional protein includes a nuclease functional domain and/or a reverse transcriptase functional domain. The nuclease functional domain creates a single-stranded nick at the DNA target site, and then uses the 3′ end of the DNA exposed by this cleavage to prime reverse transcription of retrotransposons, in which transcribed RNA serves as template for reverse transcription. Finally, the insertion at the target site is achieved by “copy-paste”.
In some embodiments, the species sources of the functional protein include Arthropoda or Chordata. In some embodiments, the species sources of the functional protein include Insecta, Actinopteri, Chondrichthyes, Testudines, Lepidosauria or Aves. In some embodiments, the species sources of the functional protein include Accipiter gentilis, Actinemys marmorata, Agelaius tricolor, Agrochola macilenta, Anagrus nilaparvatae, Andrena haemorrhoa, Anoplius nigerrimus, Artemisiospiza belli, Asobara japonica, Athalia rosae, Blastobasis lacticolella, Bombus hortorum, Bombus hypnorum, Bombus pratorum, Bombus vancouverensis, Brenthis ino, Cardiocondyla obscurior, Cerceris rybyensis, Clusia tigrina, Colaptes auratus, Crematogaster levior, Crotalus tigris, Cuora mccordi, Dinocampus coccinellae, Dolichovespula saxonica, Drosophila albomicans, Drosophila saltans, Ennomos quercinarius, Eopsaltria australis, Eristalis pertinax, Erithacus rubecula, Euphyes dion, Heliconius hecale, Hesperophylax magnus, Ichneumon xanthorius, Jera tricuspidata, Junonia litoralis, Lasioglossum baleicum, Lasioglossum lativentre, Lasioglossum morio, Leptopilina heterotoma, Lysandra coridon, Marasmarcha lunaedactyla, Mimumesa dahlbomi, Muschampia plurimacula, Neodiprion fabricii, Neodiprion pinetum, Nephrotoma flavescens, Nomada fabriciana, Nylanderia fulva, Nymphalis io, Papilio machaon, Pararge aegeria, Podocnemis expansa, Poecilia wingei, Prinia subflava, Scaptomyza hsui, Schistocerca americana, Seladonia tumulorum, Sphecodes monilicornis, Triplophysa tibetana, Trypoxylus dichotomus, Urbanus tucuti, Venturia canescens, Vespa crabro, Zaprionus camerounensis, or Zaprionus kolodkinae.
According to an embodiment of the present application, a nucleic acid can be provided, wherein, the nucleic acid encodes the functional protein described in the present application.
According to an embodiment of the present application, a nucleic acid set can be provided, the nucleic acid set includes a 5′-untranslated region, wherein, the 5′-untranslated region includes at least one of the nucleotide sequences as shown in SEQ ID NO: 95-188.
According to an embodiment of the present application, a nucleic acid set can be provided, the nucleic acid set includes a 3′-untranslated region, wherein, the 3′-untranslated region includes at least one nucleotide sequence as shown in SEQ ID NO: 189-282.
According to an embodiment of the present application, a nucleic acid set can be provided, the nucleic acid set includes a 5′-untranslated region and a 3′-untranslated region, wherein, the 5′-untranslated region includes the nucleotide sequence as shown in any one of SEQ ID NO: 95-188 or a variant thereof, and the 3′-untranslated region includes the nucleotide sequence as shown in any one of SEQ ID NO: 189-282 or a variant thereof, the RNA transcribed from the nucleic acid set can bind to the functional protein encoded by a specific retrotransposon. In some embodiments, the RNA transcribed from the untranslated region can recognize and bind to the RNA binding domain of the functional protein encoded by the specific retrotransposon.
According to an embodiment of the present application, a nucleic acid construct can be provided, the nucleic acid construct includes the nucleic acid encodes the functional protein described in the present application and/or the nucleic acid set described in the present application. In some embodiments, the nucleic acid construct further includes an exogenous nucleic acid fragment. In some embodiments, the exogenous nucleic acid fragment is operably inserted into the nucleic acid construct through a polyclonal insertion site, the exogenous nucleic acid fragment may be one or more, and may be the same or different. In some embodiments, the exogenous nucleic acid fragment includes any gene of interest or any gene that is transposable, preferably, the exogenous nucleic acid fragment includes a gene of a natural functional protein, an artificial chimeric gene, and/or a gene of a non-coding RNA. In some embodiments, the gene of a non-coding RNA includes a variety of RNAs with known functions and RNAs with unknown functions, such as rRNA, tRNA, small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), and/or long non-coding RNA (lncRNA). In some embodiments, the artificial chimeric gene includes a gene of a chimeric antigen receptor. In some embodiments, the gene of a natural functional protein includes a fluorescence-based reporter gene, a luciferase gene, and/or an antibiotic resistance gene. In some non-limiting embodiments, the exogenous nucleic acid fragment may further include a primer series for polymerase chain reaction to facilitate screening for retrotransposition activity.
In some embodiments, the nucleic acid construct further optionally includes a homologous sequence having 100% identity with at least 10 nucleotides of a specific region in the cell genome, preferably, further optionally includes a homologous sequence having 100% identity with at least 10 nucleotides of the gene encoding 28s rRNA. In some non-limiting embodiments, a part of the homologous sequence is identity to the target site to facilitate the initiation of the reverse transcription process. In some non-limiting embodiments, the nucleic acid construct includes 5′-untranslated region and 3′-untranslated region described in the present application, and the homologous sequences are upstream of 5′-untranslated region and/or downstream of 3′-untranslated region. In some non-limiting embodiments, the homologous sequence is upstream of 5′-untranslated region. In some non-limiting embodiments, the homologous sequence is downstream of 3′-untranslated region. In some non-limiting embodiments, the homologous sequences are upstream of 5′-untranslated region and downstream of 3′-untranslated region.
In some embodiments, the nucleic acid construct further includes a promoter and a poly (A) sequence. The promoter can be any suitable promoter sequence, that is, a nucleic acid sequence that can be recognized by a host cell expressing the exogenous nucleic acid fragment. The promoter sequence contains a transcriptional regulatory sequence that mediates the expression of the protein or polypeptide. The promoter can be any nucleic acid sequence having transcriptional activity in a selected host cell, including mutant, truncated and heterozygous promoters, and can be derived from genes encoding extracellular or intracellular proteins or polypeptides homologous or heterologous to the host cell. In some embodiments, the promoter includes CMV, EF1a, SV40, PGK, UbC, human beta actin, CAG, TRE, UAS, Ac5, GFAP, Polyhedrin promotor, TBG, ALB, ApoEHCR-hAAT, CaMKIIa, GAL1, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, U6, T7, T7lac, Sp6, araBAD, trp, lac, Ptac, or pL. Poly (A) tailing signal sequences well known in the art, as well as various truncated forms of poly (A) tailing signals, can be used in the present application.
In some embodiments, the nucleic acid construct further includes any transcription termination sequence (i.e., a sequence that is recognized by the host cell to terminate transcription) to control the expression of the exogenous nucleic acid fragment. Any terminator that is functional in the host cell of choice can be used in the present application.
Optionally, the nucleic acid construct may further include a suitable leader sequence (i.e., an untranslated region in the mRNA that is important for translation in the host cell) to control the expression of the exogenous nucleic acid fragment. The leader sequence is operably linked to the 5′-terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice can be used in the present application.
Optionally, the nucleic acid construct may further include a propeptide coding region to control the expression of the exogenous nucleic acid fragment, the propeptide coding region encodes an amino acid sequence located at the amino terminus of the polypeptide. The resulting polypeptide is called a zymogen or propolypeptide. The propolypeptide is usually inactive and can be converted into a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
Optionally, the nucleic acid construct may further include a regulatory sequence that can regulate the expression of the exogenous nucleic acid fragment according to the growth conditions of the host cell. Examples of the regulatory sequence are systems that turn gene expression on or off in response to chemical or physical stimuli, including in the presence of regulatory compounds. Other examples of the regulatory sequence are those that enable gene amplification. In these instances, the exogenous nucleic acid fragment should be operably linked to the regulatory sequence.
According to an embodiment of the present application, a composition may be provided, wherein, the composition includes: a functional protein or a functional fragment thereof encoded by a R2 family retrotransposon, or a nucleic acid encoding the functional protein or the functional fragment thereof, the functional protein or the functional fragment thereof has the function of catalyzing the insertion of an exogenous nucleic acid fragment into the genome of a cell; and a nucleic acid set, the RNA transcribed by the nucleic acid set can be recognized by a functional protein or a functional fragment thereof encoded by a specific retrotransposon.
In some embodiments, the composition is selected from at least one of the following groups (1) to (95), and any one of the following groups (1) to (94) includes: a functional protein-related sequence and a nucleic acid set,
In some embodiments, the nucleic acid set further includes an exogenous nucleic acid fragment. In some embodiments, the exogenous nucleic acid fragment is operably inserted into the nucleic acid construct through a polyclonal insertion site, the exogenous nucleic acid fragment may be one or more, and may be the same or different. In some embodiments, the exogenous nucleic acid fragment includes any gene of interest or any gene that is transposable, preferably, the exogenous nucleic acid fragment includes a gene of a natural functional protein, an artificial chimeric gene, and/or a gene of a non-coding RNA. In some embodiments, the gene of a non-coding RNA includes a variety of RNAs with known functions and RNAs with unknown functions, such as rRNA, tRNA, small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), and/or long non-coding RNA (lncRNA). In some embodiments, the artificial chimeric gene includes a gene of a chimeric antigen receptor. In some embodiments, the gene of a natural functional protein includes a fluorescence-based reporter gene, a luciferase gene, and/or a resistance gene. In some non-limiting embodiments, the exogenous nucleic acid fragment may further include a primer series for polymerase chain reaction to facilitate screening for retrotransposition activity.
In some embodiments, the nucleic acid set further optionally includes a homologous sequence having 100% identity with at least 10 nucleotides of a specific region in the cell genome of, preferably, further optionally includes a homologous sequence having 100% identity with at least 10 nucleotides of the gene encoding 28s rRNA. In some non-limiting embodiments, a part of the homologous sequence are identity to the target site to facilitate the initiation of the reverse transcription process. In some non-limiting embodiments, the nucleic acid construct includes 5′-untranslated region and 3′-untranslated region described in the present application, and the homologous sequences are upstream of 5′-untranslated region and/or downstream of 3′-untranslated region. In some non-limiting embodiments, the homologous sequence is upstream of 5′-untranslated region. In some non-limiting embodiments, the homologous sequence is downstream of 3′-untranslated region. In some non-limiting embodiments, the homologous sequences are upstream of 5′-untranslated region and downstream of 3′-untranslated region.
In some embodiments, the nucleic acid and/or nucleic acid set further includes a promoter and a poly (A) sequence. The promoter can be any suitable promoter sequence, that is, a nucleic acid sequence that can be recognized by a host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates the expression of the protein or polypeptide. The promoter can be any nucleic acid sequence having transcriptional activity in a selected host cell, including mutant, truncated and heterozygous promoters, and can be derived from genes encoding extracellular or intracellular proteins or polypeptides homologous or heterologous to the host cell. In some embodiments, the promoter includes CMV, EF1a, SV40, PGK, UbC, human beta actin, CAG, TRE, UAS, Ac5, GFAP, Polyhedrin promotor, TBG, ALB, ApoEHCR-hAAT, CaMKIIa, GAL1, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, U6, T7, T7lac, Sp6, araBAD, trp, lac, Ptac, or pL. Poly (A) tailing signal sequences well known in the art, as well as various truncated forms of poly (A) tailing signals, can be used in the present application.
In some embodiments, the nucleic acid encoding the amino acid sequence and/or the nucleic acid set further includes any transcription termination sequence, i.e., a sequence that is recognized by the host cell to terminate transcription. The termination sequence is operably linked to the 3′-terminus of the nucleic acid sequence encoding the protein or polypeptide. Any terminator that is functional in the host cell of choice can be used in the present application.
Optionally, the nucleic acid encoding the amino acid sequence and/or the nucleic acid set may further include a suitable leader sequence, i.e., an untranslated region in the mRNA that is important for translation in the host cell. The leader sequence is operably linked to the 5′-terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice can be used in the present application.
Optionally, the nucleic acid encoding the amino acid sequence and/or the nucleic acid set may further include a propeptide coding region, which encodes an amino acid sequence located at the amino terminus of the polypeptide. The resulting polypeptide is called a zymogen or propolypeptide. The propolypeptide is usually inactive and can be converted into a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
Optionally, the nucleic acid encoding the amino acid sequence and/or the nucleic acid set may further include a regulatory sequence that can regulate the expression of the polypeptide according to the growth conditions of the host cell. Examples of the regulatory sequence are systems that turn gene expression on or off in response to chemical or physical stimuli, including in the presence of regulatory compounds. Other examples of the regulatory sequence are those that enable gene amplification. In these instances, the nucleic acid sequence encoding the protein or polypeptide should be operably linked to the regulatory sequence.
According to an embodiment of the present application, a recombinant vector can be provided, wherein, the recombinant vector includes the nucleic acid encoding the functional protein described in the present application, the nucleic acid described in the present application, the nucleic acid set described in the present application, the nucleic acid construct described in the present application, or the composition described in the present application. The recombinant vector can be any suitable vector. In some embodiments, the recombinant vector includes, but is not limited to, a recombinant cloning vector, a recombinant eukaryotic expression plasmid, or a recombinant viral vector. In some embodiments, the recombinant eukaryotic expression plasmid includes pcDNA3.1, pCMV, pUC18, pUC19, pUC57, pBAD, pET, pENTR, pGenlenti, or pAAV. In some embodiments, the recombinant virus vector includes a recombinant adenovirus vector, a recombinant adeno-associated virus vector, a recombinant retrovirus vector, a recombinant herpes simplex virus vector, or a recombinant vaccinia virus vector. The recombinant vector of the present application can be constructed using methods well known in the art. For example, depending on the restriction sites contained in the backbone vector used, appropriate restriction sites can be added to both ends of the nucleic acid construct of the present application, and then loaded into the backbone vector.
According to an embodiment of the present application, a recombinant host cell can be provided, wherein, the recombinant host cell comprises the functional protein described in the present application, the nucleic acid encoding the functional protein described in the present application, the nucleic acid described in the present application, the nucleic acid set described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, or the recombinant vector described in the present application. The recombinant host cell can be any host cell in which retrotransposons can be used. In some embodiments, the recombinant host cell includes, but is not limited to, an animal cell, a plant cell, an algal cell, a fungal cell, a yeast cell, or a bacterial cell. In some embodiments, the animal cell includes a mammalian cell. In some embodiments, the mammalian cell includes a primary cell (e.g., a mesenchymal stem cell, an endothelial cell, an epithelial cell, a fibroblast, a keratinocyte, a melanocyte, a smooth muscle cell, and an immune cell), an immortalized cell line (e.g., HEK293, NIH-3T3, RAW-264.7, STO, VERO, CT26, hTERT immortalized human endothelial/epithelial/fibroblast/keratinocyte/ductal/cell lines), a cancer cell line (e.g., Hela, HepG2/3, HL-60, HT-1080, HT-29, A549, SW620, HCT-15, HCT116, MDA-MB-231, MCF7, SK-OV-3, PANC-1, AsPc-1, THP-1, Huh7, KG-1, RAJI, HB-CB, Jurkat, K562, CRL5826, CHO, MDCK, and Renca), an embryonic stem cell line (e.g., H1, H9, WIBR2, WIBR3, G-Olig2, ESF158, RW.4. R1, and D3) and differentiated cells thereof, or an induced pluripotent stem cell line and differentiated cells thereof.
According to an embodiment of the present application, a kit can be provided, wherein, the kit includes the functional protein described in the present application, the nucleic acid encoding the functional protein described in the present application, the nucleic acid described in the present application, the nucleic acid set described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, the recombinant vector described in the present application, or the recombinant host cell described in the present application.
The retrotransposon-based tools and methods for large fragment gene insertion and integration provided in the present application can be applied to many fields such as gene therapy, crop breeding, model animal engineering, and industrial microorganism engineering. Especially in the field of gene therapy and cell therapy, the tools and methods can be applied to gene writing therapy, which is of great significance for the treatment of genetic diseases that require long fragment gene correction.
According to an embodiment of the present application, a method for introducing an exogenous nucleic acid fragment into the genome of a host cell can be provided, wherein, the method comprises: delivering the functional protein described in the present application, the nucleic acid encoding the functional protein described in the present application, the nucleic acid described in the present application, the nucleic acid set described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, or the recombinant vector described in the present application into the host cell.
According to an embodiment of the present application, a method for editing the genome of a host cell can be provided, wherein, the method comprises: delivering the functional protein described in the present application, the nucleic acid encoding the functional protein described in the present application, the nucleic acid described in the present application, the nucleic acid set described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, or the recombinant vector described in the present application into the host cell.
According to an embodiment of the present application, a method for obtaining a host cell containing an exogenous nucleic acid fragment in the genome can be provided, wherein, the method comprises: delivering the functional protein described in the present application, the nucleic acid encoding the functional protein described in the present application, the nucleic acid described in the present application, t the nucleic acid set described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, or the recombinant vector described in the present application into the host cell.
The method of delivery into the host cell can be any suitable method. In some embodiments, the delivery method includes but is not limited to cationic liposome delivery, lipoid nanoparticle delivery, cationic polymer delivery, vesicle-exosome delivery, gold nanoparticle delivery, polypeptide and protein delivery, retrovirus delivery, lentivirus delivery, adenovirus delivery, adeno-associated virus delivery, electroporation delivery, agrobacterium infection delivery, or gene gun delivery. The methods of cell transfection and culture are routine methods in the art, and appropriate transfection and culture methods can be selected according to different cell types.
The host cell can be any host cell in which retrotransposons can be used. In some embodiments, the host cell includes, but is not limited to, an animal cell, a plant cell, an algal cell, a fungal cell, a yeast cell, or a bacterial cell. In some embodiments, the host cell includes a mammalian cell. In some embodiments, the host cell includes a primary cell (e.g., a mesenchymal stem cell, an endothelial cell, an epithelial cell, a fibroblast, a keratinocyte, a melanocyte, a smooth muscle cell, and an immune cell), an immortalized cell line (e.g., HEK293, NIH-3T3, RAW-264.7, STO, VERO, CT26, hTERT immortalized human endothelial/epithelial/fibroblast/keratinocyte/ductal/cell lines), a cancer cell line (e.g., Hela, HepG2/3, HL-60, HT-1080, HT-29, A549, SW620. HCT-15, HCT116, MDA-MB-231, MCF7, SK-OV-3, PANC-1, AsPc-1, THP-1, Huh7, KG-1, RAJI, HB-CB, Jurkat, K562, CRL5826, CHO, MDCK, and Renca), an embryonic stem cell line (e.g., H1, H9, WIBR2, WIBR3, G-Olig2, ESF158, RW.4, R1, and D3) and differentiated cells thereof, or an induced pluripotent stem cell line and differentiated cells thereof.
According to an embodiment of the present application, use of the functional protein described in the present application, the nucleic acid encoding the functional protein described in the present application, the nucleic acid described in the present application, the nucleic acid set described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, the recombinant vector described in the present application, or the recombinant host cell described in the present application for introducing an exogenous nucleic acid fragment into the genome of a host cell can be provided. The host cell can be any host cell in which retrotransposons can be used. In some embodiments, the host cell includes, but is not limited to, an animal cell, a plant cell, an algal cell, a fungal cell, a yeast cell, or a bacterial cell. In some embodiments, the host cell includes a mammalian cell. In some embodiments, the host cell includes a primary cell (e.g., a mesenchymal stem cell, an endothelial cell, an epithelial cell, a fibroblast, a keratinocyte, a melanocyte, a smooth muscle cell, and an immune cell), an immortalized cell line (e.g., HEK293, NIH-3T3, RAW-264.7, STO, VERO, CT26, hTERT immortalized human endothelial/epithelial/fibroblast/keratinocyte/ductal/cell lines), a cancer cell line (e.g., Hela, HepG2/3, HL-60, HT-1080, HT-29, A549, SW620. HCT-15, HCT116, MDA-MB-231, MCF7, SK-OV-3, PANC-1, AsPc-1, THP-1, Huh7, KG-1, RAJI, HB-CB, Jurkat, K562, CRL5826, CHO, MDCK, and Renca), an embryonic stem cell line (e.g., H1, H9, WIBR2, WIBR3, G-Olig2, ESF158, RW.4, R1, and D3) and differentiated cells thereof, or an induced pluripotent stem cell line and differentiated cells thereof.
According to an embodiment of the present application, use of the functional protein described in the present application, the nucleic acid encoding the functional protein described in the present application, the nucleic acid described in the present application, the nucleic acid set described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, the recombinant vector described in the present application, or the recombinant host cell described in the present application for gene therapy, cell therapy, genomic research, or stem cell induction and post-induction differentiation can be provided.
The above various embodiments and preferences for the present application can be combined with each other (as long as they are not inherently contradictory to each other and are suitable for the use of the present application), and the various embodiments formed by such combinations are considered as a part of the present application.
Exemplary embodiments of the present application are described below in conjunction with the accompanying drawings, where various details of the examples of the present application are included to facilitate understanding. It should be understood that they are considered to be exemplary only and not intended to limit the protection scope of the present application. The protection scope of the present application is only defined by the claims. Therefore, those of ordinary skill in the art should be aware that various changes and modifications can be made to the examples described herein, without departing from the scope of the present application. Likewise, for clarity and conciseness, the description of well-known functions and structures is omitted in the following description.
Unless otherwise stated, the reagents and instruments used in the following examples are conventional products that are commercially available. Unless otherwise stated, experiments are performed under conventional conditions or conditions recommended by the manufacturer.
We designed a high-throughput screening system to detect the insertion activity of different retrotransposons of the present application at specific sites in the 28s rRNA gene of the human genome.
The order of the elements in the vector is shown in
Secondly, based on the functional characteristics of the R2 retrotransposon, we added sequences (R105, R100 in
In order to use a unified method to detect the retrotransposition activity of all retrotransposons, we inserted a consensus sequence (universal seq) between the ORF region encoding a functional protein and the 3′ UTR, which contained a PCR primer F1 (with a sequence as shown in SEQ ID NO: 301, 5′-TGTGCCGAGGCTCAGGCACGCTC-3′) with high specificity and amplification efficiency in this sequence. After the retrotransposition process was completed and R2 was inserted into the 28s rRNA gene, the genomic DNA was extracted and used as a template to amplify the junction of 28s rRNA gene with retrotransposons using F1 and the reverse primer al (with a sequence as shown in SEQ ID NO: 302, 5 ‘-GGCCTCCCACTTATTCTACACC-3’), about 200 bp downstream of the insertion site. PCR products of different lengths would be amplified for different R2 samples due to differences in the length of their 3′ UTR sequences. The products were further analyzed by Sanger sequencing to verify whether directional insertion at the 28s rRNA gene locus was achieved.
After HEK293T cells (commercially purchased) were cultured to the logarithmic growth phase, they were digested and dispersed into single cells with 0.25% Trypsin (Thermo), and added to a 96-well cell culture plate pre-coated with PDL (Sigma) at a cell concentration of 1×104 cells/well, and cultured overnight at 37° C. in 5% CO2.
A dosage of 200 ng of each retrotransposon plasmid constructed in example 1 was mixed with the transfection reagent Lipofectamine 2000 (Thermo) according to the ratio of transfection plasmid mass (μg): transfection reagent volume (μL)=1:2, and allowed to stand at room temperature for 15 minutes to form a transfection complex. The transfection complex was transferred to the cell culture plate and incubated with the cells, and two parallel tests were performed for each sample to be screened.
72 hours after transfection, the cells were digested and dispersed into single cells with 0.25% Trypsin (Thermo). The cells were collected by centrifugation and the genomic DNA of the cells was extracted using a genome extraction kit (BEIJING BIOTEKE BIOTECHNOLOGY CO., LTD, DP1202), and the concentration of the genomic DNA was measured using a nanodrop one ultramicrovolume spectrophotometer (Thermo).
The genomic DNA of all samples was subjected to PCR analysis according to the conditions in Table 2 and Table 3, and the PCR products were detected by 1% agarose gel electrophoresis.
The results of retrotransposition activity are shown in
Using a human single-copy gene 36b4 as an internal reference gene, a relative quantitative analysis of the retrotransposition activities of some retrotransposons in Table 1 was performed. The method was the same as that described in examples 1 and 2, the only difference was that: during the PCR process, an additional PCR primer pair targeting the single-copy gene 36b4 was added at the same time:
The results of retrotransposition activity are shown in
The method used to detect the 5′ junction of 28s rRNA gene with the retrotransposons in Table 1, which showed the integrity of retrotransposition event. The method is the same as that described in examples 1 and 2, the only difference was that: 3 reverse primers were designed for different positions in the transposon ORF region and 5′ UTR sequence (the sequences of the reverse primers are shown in Table 4), and paired with the forward primer (28s-up-F1, with a sequence as shown in SEQ ID NO: 305), about 200 bp upstream of the insertion site in 28s rRNA gene for PCR analysis, respectively. The integrity of the 5′ end sequences of these samples after insertion was detected to preliminarily determine the retrotransposition integrity of different R2 samples.
Retrotransposition integrity assay results are shown in
It should be stated that the above are only the preferred examples of the present application and are not intended to limit the present application. For those of ordinary skill in the art, various modifications and changes can be made to the present application. Although the specific embodiments have been described, for the applicant or a person skilled in the art, the substitutions, modifications, changes, improvements, and substantial equivalents of the above embodiments may exist or cannot be foreseen currently. Therefore, the submitted appended claims and claims that may be modified are intended to cover all such substitutions, modifications, changes, improvements, and substantial equivalents. It is important that, as the technology evolves, many elements described herein may be replaced with equivalent elements that appear after the present application.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202310380978.4 | Apr 2023 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2024/081116 | 3/12/2024 | WO |
