KR20210121756A - Safe retroviral vectors developed by insertion of histone or leucine zipper into the viral integrase - Google Patents

Abstract

The present invention relates to safe retroviral vectors developed through introduction of histone or leucine zipper into integrase, a method for developing the same, and a use thereof. The present invention inserts histone or leucine zipper in front of the 37^th and 273^rd residues of an integrase gene to extinct integration preference with respect to a transcriptional start site (TSS) so that risk of overexpressing oncogenes is removed, thereby being usefully used for clinical application and disease treatment.

Description

{Safe retroviral vectors developed by insertion of histone or leucine zipper into the viral integrase}

The present invention relates to a retroviral vector constructed through the introduction of histone or leucine zipper into an integrase, a method for constructing the same, and a use thereof.

Retroviral vectors can carry large gene units of up to 8 kb and do not elicit a specific immune response. Also, unlike other types of viral vectors, retroviral vectors enable long-term gene expression through integration of therapeutic genes into the human genome.

Murine leukemia virus (MLV), which has been studied for a long time, was used as a platform for developing retroviral vectors. Retroviral vectors include X-linked severe combined immunodeficiency (SCID-X1), adenosine deaminase deficiency-severe combined immunodeficiency (ADA-SCID) and Wiskott- It has been used to treat genetic disorders such as Aldrich syndrome (Wiskott-Aldrich syndrome; WAS). Such an attempt has resulted in an almost complete therapeutic effect on the patient.

Recently, Strimvelis, an MLV vector-based gene therapy drug for the treatment of ADA-SCID patients, was approved in Europe. Streambellis is a therapy using CD34+ cells into which a retroviral vector carrying a gene encoding human adenosine deaminase has been introduced.

In addition, there are two approved retroviral-based therapies. Zalmoxis is a retroviral vector carrying a gene encoding a truncated form of human low affinity nerve growth factor receptor (ΔLNGFR) and a mutated herpes simplex type I virus thymidine kinase (HSV-TK Mut2). Based on transduced allogeneic T cells (HSV-TK Mut2). Yescarta is a therapy consisting of T cells engineered to express a chimeric antigen receptor on the surface by applying a retroviral vector.

In contrast to their positive properties for therapeutic use, MLV-based retroviral vectors have carcinogenic potential. These adverse properties have been validated experimentally and clinically. The carcinogenic potential of MLV is related to MLV's preference for regions of the human genome involved in gene expression regulation. In particular, when a retrovirus is inserted into a genomic site near the oncogene TSS, the gene can be activated, from which a cell having an integrated retrovirus can be transformed into a cancer cell. This MLV-specific, specific human genome segment insertion preference limits its widespread use as a vector platform in clinical applications.

It has recently been reported that the pattern of MLV insertion into the human genome is partly related to the interaction of viral integrase with bromodomain and extra-terminal (BET) family proteins. Based on these findings, several attempts have been made to block the relevant interaction by mutating the viral integrase or introducing small molecules (which selectively interfere with the binding of BET proteins to chromosomes) into cells. These methods can reduce MLV insertion into regulatory genomic domains, including TSS. However, the frequency of virus insertion into the region to be controlled is still much higher than the random probability. These results indicate that additional host factors or several unknown mechanisms contribute to the human genome-specific region insertion preference of MLV.

Another type of retrovirus, human immunodeficiency virus (HIV-1), is known to follow a mechanism that induces the insertion of epithelial-derived growth factor (LEDGF/p75) into transcriptional units. However, it was also observed that removal of the gene encoding LEDGF/p75 could not completely block HIV-1 insertion into the transcription unit. Discovered that additional host factors (hepatoma-derived growth factor-related protein 2 [HRP-2]) also direct viral pro-integration complexes to specific preferred genomic regions show that with limited knowledge of the host factors involved in the determination of the viral insertion site, it is difficult to completely switch the HIV-1 insertion pattern.

Since it is impossible to completely block the action of unknown host factors, we instead attempted to alter the retroviral gene insertion pattern by perturbing the retroviral integrase structure.

The present inventors changed the gene insertion pattern of the MLV-based retroviral vector favoring TSS by inserting histone and leucine zippers into the internal sites of integrase. As a result, a gene insertion pattern in which the preference for TSS was lowered to a random level was obtained.

In addition, the integrase mutation greatly reduced the frequency of retroviral insertion into the oncogenic region. From the predicted structural comparison of wild-type and mutant integrase, it was inferred that the altered retrovirus gene insertion pattern was related to structural changes in some subdomains of the enzyme, presumed to be due to the insertion of histone and leucine zippers.

It was hypothesized that the structural modification of integrase was partially related to the newly formed hydrogen bond between the inserted protein and the subdomain of the corresponding viral protein. The results obtained by the inventors show that elaborately changing the integrase structure is a good way to construct a safe retroviral vector.

Accordingly, the present inventors introduced histone or leucine zipper proteins into MLV specific internal sites to change the retroviral gene insertion pattern. Histones can interact with other types of histones and leucine zippers can dimerize with other leucine zippers. Based on these characteristics of the two types of proteins, the present inventors confirmed that when the corresponding proteins were introduced into the viral integrase, a large change occurred in the MLV gene insertion pattern.

Accordingly, it is an object of the present invention to provide a retroviral vector constructed through the introduction of histone or leucine zipper into integrase.

Another object of the present invention is to provide a method for constructing a retroviral vector through the introduction of a histone or leucine zipper into an integrase.

Another object of the present invention is to provide the use of a retroviral vector constructed through the introduction of a histone or leucine zipper into an integrase.

The present invention relates to a method for constructing a safe retroviral vector through the introduction of histone or leucine zipper into an integrase.

The present invention introduces a histone or leucine zipper before the 37th and/or 273th residue of the integrase protein to eliminate the integration preference for a transcriptional start site (TSS), so that the constructed vector is an oncogene It will eliminate the risk of overexpression and make it useful for clinical applications and disease treatment.

Hereinafter, the present invention will be described in more detail.

An example of the present invention relates to introducing a gene encoding a histone or leucine zipper into a gene for Gag-Pol expression, which is a major element of a retroviral vector.

In the present invention, the retroviral vector may be an MLV-based retroviral vector, an HIV-1 based retroviral vector, or an ASLV-based retroviral vector, for example, it may be an MLV-based retroviral vector, but is limited thereto. it's not going to be

In the present invention, the gene encoding a histone or leucine zipper may be introduced into a gene encoding an integrase.

In the present invention, the integrase may be MLV integrase, HIV-1 integrase, ASLV integrase, etc., for example, may be MLV integrase, but is not limited thereto.

In the present invention, the gene encoding integrase may include the nucleotide sequence of SEQ ID NO: 83, for example, it may consist of the nucleotide sequence of SEQ ID NO: 83, but is not limited thereto.

In the present invention, integrase may include the amino acid sequence of SEQ ID NO: 84, for example, may consist of the amino acid sequence of SEQ ID NO: 84, but is not limited thereto.

In the present invention, the histone may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 70 to 76, for example, may consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 70 to 76, but is limited thereto it is not

In the present invention, the histone-encoding gene may include a nucleotide sequence selected from the group consisting of SEQ ID NOs: 52 to 58, for example, may consist of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 52 to 58 , but is not limited thereto.

In the present invention, the histone may include the first linker at the C-terminus, the N-terminus or both ends, but is not limited thereto.

In the present invention, the gene encoding the histone may include a gene encoding the first linker in 3', 5' or both, but is not limited thereto.

In the present invention, any linker used by a person skilled in the art may be used as the first linker.

In one embodiment of the present invention, the first linker may include the amino acid sequence of SEQ ID NO: 77, for example, may consist of the amino acid sequence of SEQ ID NO: 77, but is not limited thereto.

In one embodiment of the present invention, the gene encoding the first linker may include the nucleotide sequence of SEQ ID NO: 59, for example, it may consist of the nucleotide sequence of SEQ ID NO: 59, but is limited thereto no.

In the present invention, the leucine zipper comprises a NZ sequence comprising the amino acid sequence of SEQ ID NO: 78 and a CZ sequence comprising the amino acid sequence of SEQ ID NO: 79; Or it may include a Jun sequence comprising the amino acid sequence of SEQ ID NO: 80 and a Fos sequence comprising the amino acid sequence of SEQ ID NO: 81, for example, the NZ sequence consisting of the amino acid sequence of SEQ ID NO: 78 and SEQ ID NO: 79 a CZ sequence consisting of an amino acid sequence; Alternatively, the Jun sequence consisting of the amino acid sequence of SEQ ID NO: 80 and the Fos sequence consisting of the amino acid sequence of SEQ ID NO: 81 may include, but are not limited thereto.

In the present invention, the gene encoding the NZ sequence may be a nucleotide sequence including the nucleotide sequence of SEQ ID NO: 60, for example, it may be a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 60, but is limited thereto no.

In the present invention, the gene encoding the CZ sequence may be a nucleotide sequence including the nucleotide sequence of SEQ ID NO: 61, for example, may be a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 61, but is limited thereto no.

In the present invention, the gene encoding the Jun sequence may be a nucleotide sequence including the nucleotide sequence of SEQ ID NO: 62, for example, it may be a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 62, but is limited thereto no.

In the present invention, the gene encoding the Fos sequence may be a nucleotide sequence including the nucleotide sequence of SEQ ID NO: 63, for example, may be a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 63, but is limited thereto no.

In the present invention, the leucine zipper may include a second linker at the C-terminus, the N-terminus or both ends, but is not limited thereto.

In the present invention, the gene encoding the leucine zipper may include a gene encoding a second linker in 3', 5', or both, but is not limited thereto.

NZ sequence and CZ sequence in the present invention; Alternatively, the Jun sequence and the Fos sequence may be linked through a second linker, but the present invention is not limited thereto.

As the second linker in the present invention, any linker used by a person skilled in the art may be used.

In one embodiment of the present invention, the second linker may include the amino acid sequence of SEQ ID NO: 82, for example, may consist of the amino acid sequence of SEQ ID NO: 82, but is not limited thereto.

In one embodiment of the present invention, the gene encoding the second linker may include one type of nucleotide sequence selected from the group consisting of SEQ ID NO: 64 to SEQ ID NO: 69, for example, SEQ ID NO: 64 to sequence It may consist of one type of nucleotide sequence selected from the group consisting of number 69, but is not limited thereto.

In one embodiment of the present invention, when the leucine zipper comprises the NZ sequence comprising the amino acid sequence of SEQ ID NO: 78 and the CZ sequence comprising the amino acid sequence of SEQ ID NO: 79, the gene encoding the leucine zipper is 5 ' may include a gene encoding a second linker comprising SEQ ID NO: 64, and 3' may include a gene encoding a second linker comprising SEQ ID NO: 66, for example, 5' includes a sequence It may include a gene encoding a second linker consisting of number 64, and may include a gene encoding a second linker consisting of SEQ ID NO: 66 in 3', but is not limited thereto.

In one embodiment of the present invention, when the leucine zipper comprises a Jun sequence comprising the amino acid sequence of SEQ ID NO: 80 and a Fos sequence comprising the amino acid sequence of SEQ ID NO: 81, the gene encoding the leucine zipper is 5 ' may include a gene encoding a second linker comprising SEQ ID NO: 67, and 3' may include a gene encoding a second linker comprising SEQ ID NO: 69, for example, 5' has a sequence It may include a gene encoding a second linker consisting of number 67, and may include a gene encoding a second linker consisting of SEQ ID NO: 69 in 3', but is not limited thereto.

In one embodiment of the present invention, when the NZ sequence and the CZ sequence are linked through a second linker, the gene encoding the second linker may include the nucleotide sequence of SEQ ID NO: 65, for example, SEQ ID NO: It may consist of a nucleotide sequence of 65, but is not limited thereto.

In one embodiment of the present invention, when the Jun sequence and the Fos sequence are linked through a second linker, the gene encoding the second linker may include the nucleotide sequence of SEQ ID NO: 68, for example, SEQ ID NO: It may consist of the nucleotide sequence of 68, but is not limited thereto.

In the present invention, the gene encoding the histone or the gene encoding the leucine zipper may be inserted after position 108 or 816 of the gene encoding integrase.

In the present invention, the histone or leucine zipper may be inserted before residues 37 or 273 of integrase. Through this, by eliminating the preference for integration to the transcriptional start site (TSS), the risk of overexpressing oncogenes is eliminated, and it can be usefully used in clinical applications and disease treatment.

As used herein, the term “vector” refers to a means for expressing a target gene in a host cell. For example, it may be a retroviral vector.

In the present invention, a recombinant vector can be typically constructed as a vector for cloning or a vector for expression. The expression vector may be a conventional vector used to express a foreign protein in plants, animals or microorganisms in the art. The recombinant vector can be constructed through various methods known in the art.

For the delivery (introduction) of a gene into a host cell for production of the vector of the present invention, a method well known in the art can be used. The method is, for example, when the host cell is a prokaryotic cell, CaCl ₂ method or electroporation method, etc. can be used, and when the host cell is a eukaryotic cell, microinjection method, calcium phosphate precipitation method, electroporation method, Liposome-mediated transfection and gene bombardment may be used, but the present invention is not limited thereto.

Another embodiment of the present invention relates to a method for constructing a retroviral vector into which a gene encoding a histone or leucine zipper is introduced, comprising the following steps.

Gene preparation step of preparing a gene encoding a histone or leucine zipper; and

An introduction step in which the gene is introduced into the retroviral integrase gene.

In the present invention, the introducing step may be introducing a gene encoding a histone or leucine zipper into a gene encoding integrase.

In the method of the present invention, the description of the histone or leucine zipper is the same as described above, and the description thereof is omitted.

In the method of the present invention, the description of the gene encoding the histone or leucine zipper is the same as described above, and thus the description thereof is omitted.

Another embodiment of the present invention relates to the use of a safe retroviral vector constructed through the introduction of a histone or leucine zipper into an integrase.

In the present invention, the use is for obtaining a therapeutic effect (gene therapy) through gene introduction into various animal and human cells, for research by introducing a target gene into various animal and human cell genomes, for developing new cells for therapeutic purposes, cancer It may be for the development of an immunotherapeutic agent for treating various diseases, including, but not limited to.

For the use of the present invention, the retroviral vector and its construction method are the same as described above, and the description thereof will be omitted.

The present invention relates to a retroviral vector constructed through the introduction of histone or leucine zipper into an integrase, a method for constructing the same, and a use thereof. The present invention inserts a histone or leucine zipper before the 37th and/or 273th residue of the integrase protein to eliminate gene integration preference for a transcriptional start site (TSS), thereby overexpressing an oncogene. It can be usefully used for clinical applications and disease treatment as it eliminates the risk of possible risks.

1 is a diagram showing the construction of a mutant MLV integrase comprising a histone or leucine zipper according to an embodiment of the present invention.
2 is a graph showing the transducing titer of a viral vector according to an embodiment of the present invention.
3 is a graph showing the frequency of retroviral vector insertion into a genomic region within 5 kb of TSSs of a protein-coding gene according to an embodiment of the present invention.
Figure 4a is a graph showing the distribution of retroviral vector insertion within 5 kb from TSS according to an embodiment of the present invention.
Figure 4b is a graph showing the distribution of retroviral vector insertion within 50 kb from TSS according to an embodiment of the present invention.
Figure 4c is a graph showing the cumulative frequency of virus insertion according to the distance from the TSS according to an embodiment of the present invention.
5 is a graph showing the frequency of insertion of a retroviral vector into an oncogenic site according to an embodiment of the present invention.
Figure 6a is a graph showing the frequency of retroviral vector insertion near the CpG island according to an embodiment of the present invention.
Figure 6b is a graph showing the frequency of retroviral vector insertion near the cis regulatory element according to an embodiment of the present invention.
7A is a graph showing the distribution of retroviral vector insertion within 5 kb from CpG islands and cis-regulatory elements according to an embodiment of the present invention.
7B is a graph showing the spatial distribution of retroviral vector insertion in the range of 50 kb in CpG islands and cis-regulatory elements according to an embodiment of the present invention.
7c is a graph showing the cumulative frequency of retroviral vector insertion according to the distance from the CpG island according to an embodiment of the present invention.
7D is a graph showing the cumulative frequency of retroviral vector insertion according to the distance from the cis-regulatory element according to an embodiment of the present invention.
8A is a diagram showing the predicted structure of wild-type and mutant integrase according to an embodiment of the present invention.
FIG. 8b is a graph showing the prediction result of hydrogen bond formation between MLV integrase and the inserted histone (H3.3) according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Experimental method

Construction of Plasmids Encoding Integrase Mutants

Genes encoding seven types of histones and two types of leucine zippers were introduced at two sites within the MLV integrase gene (corresponding to amino acid residues 37 and 273 of the corresponding protein), resulting in mutations as part of the gag-pol polyprotein A plasmid encoding the chain integrase was constructed.

To obtain DNA encoding histones, cDNA synthesis was performed from mRNA of H2A (H2A.1 and H2A.2), H2B and H4 of human embryonic kidney (HEK) 293T cells, and H3 of HeLa cells cDNA synthesis was performed from mRNA (H3.1, H3.2, and H3.3). cDNA synthesis was performed according to the protocol of the SuperScript III CellsDirect cDNA synthesis system (Invitrogen, Carlsbad, CA) with oligo dT and random hexamers. Polymerase chain reaction (PCR)-based cDNA amplification was performed with the following thermal cycle:

Initial denaturation at 95 °C for 2 min;

40 cycles of denaturation at 95° C. for 30 sec, annealing at 55° C. for 30 sec and extension at 72° C. for 27 sec;

final extension at 72° C. for 2 min; and

Cool for 3 min at 4 °C.

PCR reactions were performed using a C1000 PCR machine (Bio-Rad, Hercules, CA, USA), Taq DNA polymerase (New England Biolabs (NEB), Ipswich, MA, USA) or Phusion High-Fidelity DNA Polymerase (NEB). and a primer having a linker sequence (SEQ ID NO: 1) was used. DNA sequences encoding two types of leucine zipper proteins (NZ-CZ and Jun-Fos) were obtained from Genescript (Hong Kong, China) and introduced after amplification after the CMV promoter in the pCMV gag-pol plasmid.

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number denomination Sequence Listing (5'-> 3') note One Linker DDPVAT 2 H2A_sense_primer AAAAAGCGCTGATCCACCGGTCGCCACCATGTCTGGACGTGGCAAGCAG 3 H2A_antisense_primer AAAAGCTAGCGGTGGCGACCGGTGGATCCTTGCCCTTAGCTTTGTGGTGG 4 H2B_sense_primer AAAAGGCGCCGATCCACCGGTCGCCACCATGCCTGAGCCAGCGAAATCC 5 H2B_antisense_primer AAAAGCTAGCGGTGGCGACCGGTGGATCCTTGGAGCTGGTGTACTTGGTAACG 6 H3.1_sense_primer AAAAAGCGCTGATCCACCGGTCGCCACCATGGCACGCACGAAGCAAAC 7 H3.1_antisense_primer AAAAGCTAGCGGTGGCGACCGGTGGATCTGCCCTCTCTCCGCGAATG 8 H3.2_sense_primer AAAAAGCGCTGATCCACCGGTCGCCACCATGGCCCGTACTAAGCAGACTGC 9 H3.2_antisense_primer AAAAGCTAGCGGTGGCGACCGGTGGATCAGCCCGCTCTCCACGGATG 10 H3.3_sense_primer AAAAAGCGCTGATCCACCGGTCGCCACCATGGCTCGTACAAAGCAGACTGCC 11 H3.3_antisense_primer AAAAGCTAGCGGTGGCGACCGGTGGATCAGCACGTTCTCCACGTATGCG 12 H4_sense_primer AAAAAGCGCTGATCCACCGGTCGCCACCATGTCCGGCAGAGGAAAGGG 13 H4_antisense_primer AAAAGCTAGCGGTGGCGACCGGTGGATCGCCTCCGAAGCCGTACAGG

cell culture

HEK 293T cells and HeLa cells were cultured at 37 ° C. and 5% CO ₂ conditions, respectively, using Modified Dulbecco's medium (Gibco Life Technologies, Carlsbad, CA) and Dulbecco's modified Eagle medium (Gibco Life Technologies), respectively. Each of these media was supplemented with 10% fetal bovine serum (Gibco Life Technologies) and 1% penicillin-streptomycin (Gibco Life Technologies).

Retrovirus vector packaging

For packaging retroviral vectors, the vector genome (pCLPIT GFP, 10 ug), wild-type gag-pol polyprotein or mutant (pCMV gag-pol, pCMV gag-pol-histone or pCMV gag-pol-leucine zipper, 6 ug) ) and a plasmid encoding the envelope protein (pcDNA IVS VSVG, 4 ug) were introduced into HEK 293T cells by a calcium phosphate-based transformation method. Transformed cells were washed twice with phosphate-buffered saline (PBS, Gibco Life Technologies) 12 hours after transformation, and further cultured in fresh medium. Cell supernatants containing packaged viral particles were obtained twice at 36 and 60 hours after transformation. The obtained cell supernatant was first filtered through a 0.45 um syringe filter and then Optima ?? Ultracentrifuge LE-80K (Beckman Coulter, Brea, CA, USA) was used for ultracentrifugation and concentration on a cushion of 20% (w/v) sucrose at 4°C. Ultracentrifugation was performed using SW28 at 24,000 rpm for 2 h. The resulting virus particle pellet was resuspended in cooled phosphate buffered saline.

Titration of retroviral vector transduced particles

To determine the transduction titer of viral vector particles, a gene encoding enhanced green fluorescent protein (eGFP) was introduced into the original viral genome. Titration of transgenic viral particles proceeded by transducing HEK 293T cells and counting eGFP-positive cells. eGFP-positive cells were transferred to FacsCanto ?? 3 days after transduction. Quantified by flow cytometry with a II flow cytometer (BD Biosciences, San Diego, CA).

Amplification of viral cell genome junctions

Wild-type and mutant retroviral vectors were transduced into HEK 293T cells. Then, the transduced cells were cultured for several days. Cell genomic DNA was then extracted from the cells using the QIAamp DNA mini kit (Qiagen, Valencia, CA, USA). The genomic DNA was then fragmented with BamHI (NEB), and the DNA fragment containing the retroviral sequence was bound to a single type of biotinylated oligonucleotide (SEQ ID NO: 14) and selectively and linearly amplified by PCR.

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number denomination Sequence Listing (5'-> 3') note 14 biotinylated oligonucleotide biotin-ATTTGTTAAAGAVAGGATATCAGTGGTCCAG

The PCR reaction conditions performed were as follows:

initial denaturation at 95 °C for 5 min;

40 cycles of denaturation at 95° C. for 1 min, annealing at 55° C. for 45 sec and extension at 72° C. for 90 sec;

final extension at 72 °C for 10 min; and

Cool at 4 °C for 5 min.

Then, the amplified DNA containing the viral sequence was selectively isolated using Dynabeads M-280 streptavidin (Thermo Fisher Scientific, Carlsbad, CA, USA). The amplified single-stranded virus-cell junction DNA was then made double-stranded (dsDNA) with dNTPs, random hexamers and Klenow enzymes. The synthesized dsDNA product was fragmented with Mse I (NEB) and ligated to a pre-annealed double-stranded linker. Using the linker and two primers complementary to the 3' LTR of the viral genome, the linker-linked virus-cell genome junction was amplified by PCR and reaction using Phusion high fidelity polymerase (NEB), respectively.

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number denomination Sequence Listing (5'-> 3') note 15 linker+ GTAATACGACTCACTATAGGGCTCCGCTTAAGGGAC 16 Linker- TAGTCCCTTAGCGGAG-NH ₂ 17 Primer_F GACTTGTGGTCTCGCTGTTCCTTGG 18 Primer_R GTAATACGACTCACTATAGGGCTCCCCGCTTAAG

Relevant PCR reaction conditions are as follows:

Initial denaturation at 98 °C for 2 min;

25 cycles of denaturation at 98° C. for 2 minutes, annealing at 55° C. for 90 seconds and extension at 72° C. for 1 minute;

final extension at 72 °C for 5 min; and

Cool at 10 °C for 3 min.

Next generation sequencing (NGS) of virus-cell junctions

For NGS analysis, adapter and index sequences were added to the amplified virus-cell genome junction DNA via PCR. Relevant PCR reaction conditions are as follows:

Initial denaturation at 98 °C for 2 min; and

25 cycles of denaturation at 98 °C for 2 min, annealing and extension in a single step at 72 °C for 90 sec, final extension at 72 °C for 5 min, and cooling at 10 °C for 3 min.

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number denomination Sequence Listing (5'-> 3') note 19 Forward primer for adapter addition TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGAGGGTCTCCTCTGAGTGATTGACTACC 20 Reverse primer for adapter addition GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACTCACTATAGGGCTCCGCTTAAGGGAC 21 Forward primer for index addition AATGATACGGCGACCACCGAGATCTACACFFFFFFFFTCGTCGGCAGCGTC 22 Reverse primer for index addition CAAGCAGAAGACGGCATACGAGATRRRRRRRRRGTCTCGTGGGCTCGG

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number denomination Sequence Listing (5'-> 3') note 23 FFFFFFFF_S502 CTCTCTAT 24 FFFFFFFF_S503 TATCCTCT 25 FFFFFFFF_S505 GTAAGGAG 26 FFFFFFFF_S506 ACTGCAT 27 FFFFFFFF_S507 AAGGAGTA 28 FFFFFFFF_S508 CTAAGCCT 29 FFFFFFFF_S510 CGTCTAAT 30 FFFFFFFF_S511 TCTCTCCG 31 FFFFFFFF_S513 TCGACTAG 32 FFFFFFFF_S515 TTCTAGCT 33 FFFFFFFF_S516 CCTAGAGT 34 FFFFFFFF_S517 GCGTAAGA 35 FFFFFFFF_S518 CTATTAAG 36 FFFFFFFF_S520 AAGGCTAT 37 FFFFFFFF_S521 GAGCCTTA 38 FFFFFFFF_S522 TTATGCGA 39 RRRRRRRR_N701 TCGCCTTA 40 RRRRRRRR_N702 CTAGTACG 41 RRRRRRRR_N703 TTCTGCCT 42 RRRRRRRR_N704 GCTCAGGA 43 RRRRRRRR_N705 AGGAGTCC 44 RRRRRRRR_N706 CATGCCTA 45 RRRRRRRR_N707 GTAGAGAG 46 RRRRRRRR_N710 CAGCCTCG 47 RRRRRRRR_N711 TGCCTCTT 48 RRRRRRRR_N712 TCCTCTAC 49 RRRRRRRR_N714 TCATGAGC 50 RRRRRRRR_N715 CCTGAGAT

Initial processing of sequencing and DNA reads using Illumina MiSeq was performed by Macrogen (Korea). Reads with an average quality score of less than 20 were filtered with PRINSEQ-lite (v 0.20.4) to consider only meaningful sequence data. For downstream analysis, we used the EMBOSS Needle (v 6.6.0.0) and Macrogen's own script to contain the virus 3' LTR sequence (SEQ ID NO: 51), under conditions that allowed a mismatch of the order of two bases or less. Only virus-cell genome junctions with Duplicate values in downstream analysis were removed. The cellular sequence finally obtained from the virus-cell genome junction was mapped to the human genome using the QuickMap tool (Gene Therapy Safety Group). Statistical significance of the difference in gene insertion patterns between wild-type and mutant vectors was performed based on Fisher's test.

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number denomination Sequence Listing (5'-> 3') note 51 viral 3´ LTR sequence GGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCA 48 bp

Prediction of wild-type and mutant integration structures

The structure of viral integrase was predicted using I-TASSER, which predicts protein structure by replication-exchange Monte Carlo simulations. The predicted structural model was evaluated based on a confidence score (C-score). If the C-score is between -5 and 2, it can be said that the predictive structure model has high reliability. Rendering of the predicted structures was performed using Pymol (DeLano Scientific, San Carlos, CA).

Results and Discussion

MLV vector loaded with integrase containing DNA binding protein inside

The present inventors expected that the integrase internal introduction of a DNA binding protein could change the gene insertion pattern of a retroviral vector by an integrative structural disturbance. Histone or leucine zippers were considered as DNA binding proteins. Histones are classified into linker histones (H1 and H5) and core histones (H2A, H2B, H3 and H4). Core histones have a structurally conserved motif, which consists of three helices of two short strap rings. These histones can interact with other histones. First, the two H2A-H2B dimers bind to the H3-H4 tetramer. Next, the octamers of the core histones interact with DNA to form nucleosomes. These interactions include polar interactions, hydrogen bonding, and non-polar interactions. Seven histones, including two to three variants of H2A and H3, were inserted into the MLV integrase ( FIG. 1 ).

Leucine zippers are regulatory proteins with unique structural properties. The zipper-like structure of a leucine zipper is formed by dimerization of two alpha-helical monomers. Leucine zippers use leucine residues to bind to DNA, through hydrophobic interactions. The present inventors have developed two types of leucine zippers [artificial (NZ-CZ (I, AD, & L, 2000)) and naturally derived Jun-Fos (Glover & Harrison, 1995)] inside MLV integrase. inserted.

Fourteen MLV mutant vectors in which the DNA binding protein was introduced into the integrase were produced. The DNA binding protein was inserted at two sites in the integrase, and there was no disruption of the 'vector transduction ability' ( FIGS. 1 and 2 ). The two sites are in the N-terminal extension domain (by residue 37) and catalytic domain (by residue 273) of MLV integrase. H3 (H3.1, H3.2 or H3.3) or leucine zippers (NZ-CZ or Jun-Fos) were introduced at the anterior position of integrase. In addition, H2A (H2A.1 or H2A.2), H2B, H3 (H3.1, H3.2 or H3.3), H4 or one of the two leucine zippers was introduced into the posterior position (Fig. 1).

order
number denomination sequence list note 52 H3.1 ATGGCACGCACGAAGCAAACAGCTCGTAAGTCCACTGGCGGCAAAGCCCCGCGCAAGCAGCTGGCCACTAAGGCGGCTCGCAAAAGCGCGCCAGCCACCGGTGGCGTGAAGAAGCCCCACCGCTACAGGCCTGGTACTGTCGCCCTCCGTGAAATCCGCCGCTATCAGAAATCGACTGAGCTACTGATTCGCAAGCTACCATTCCAGCGTCTGGTACGTGAGATCGCGCAGGACTTCAAGACCGACCTGCGCTTCCAGAGCTCGGCTGTGATGGCGCTGCAGGAGGCCTGCGAGGCTTACCTGGTGGGGCTCTTTGAGGACACCAACCTGTGTGCTATCCACGCCAAGCGAGTGACTATCATGCCCAAGGACATCCAGCTCGCTCGCCGCATTCGCGGAGAGAGGGCA 53 H3.2 ATGGCCCGTACTAAGCAGACTGCTCGCAAGTCGACCGGCGGCAAGGCCCCGAGGAAGCAGCTGGCCACCAAGGCGGCCCGCAAGAGCGCGCCGGCCACGGGCGGGGTGAAGAAGCCGCACCGCTACCGGCCCGGCACCGTAGCCCTGCGGGAGATCCGGCGCTACCAGAAGTCCACGGAGCTGCTGATCCGCAAGCTGCCCTTCCAGCGGCTGGTACGCGAGATCGCGCAGGACTTTAAGACGGACCTGCGCTTCCAGAGCTCGGCCGTGATGGCGCTGCAGGAGGCCAGCGAGGCCTACCTGGTGGGGCTGTTCGAAGACACGAACCTGTGCGCCATCCACGCCAAGCGCGTGACCATTATGCCCAAGGACATCCAGCTGGCCCGCCGCATCCGTGGAGAGCGGGCT 54 H3.3 ATGGCTCGTACAAAGCAGACTGCCCGCAAATCGACCGGTGGTAAAGCACCCAGGAAGCAACTGGCTACAAAAGCCGCTCGCAAGAGTGCGCCCTCTACCGGAGGGGTGAAGAAACCTCATCGTTACAGGCCTGGTACTGTGGCGCTCCGTGAAATTAGACGTTATCAGAAGTCCACTGAACTTCTGATTCGCAAACTTCCCTTCCAGCGTCTGGTGCGAGAAATTGCTCAGGACTTTAAAACAGATCTGCGCTTCCAGAGCGCAGCTATCGGTGCTTTGCAGGAGGCAAGTGAGGCCTATCTGGTTGGCCTTTTTGAAGACACCAACCTGTGTGCTATCCATGCCAAACGTGTAACAATTATGCCAAAAGACATCCAGCTGGCACGCCGCATACGTGGAGAACGTGCT 55 H2A.1 ATGTCTGGACGTGGCAAGCAGGGCGGCAAGGCTCGCGCCAAGGCCAAAACCCGCTCCTCTAGAGCTGGGCTCCAATTTCCTGTAGGACGAGTGCACCGCCTGCTCCGCAAGGGCAACTACGCTGAGCGGGTCGGGGCCGGCGCGCCGGTTTACCTGGCGGCGGTGCTGGAGTACCTAACTGCCGAGATCCTGGAGCTGGCGGGCAACGCAGCCCGCGACAACAAAAAGACCCGCATCATCCCGCGCCACTTGCAGCTGGCCATCCGCAACGACGAGGAGCTCAACAAGCTGCTTGGTAAAGTTACCATCGCTCAGGGCGGTGTTCTGCCTAACATCCAGGCCGTACTGCTCCCCAAGAAGACTGAGAGCCACCACAAAGCTAAGGGCAAG 56 H2A.2 ATGTCTGGTCGTGGCAAGCAAGGAGGCAAGGCCCGCGCCAAGGCCAAGTCGCGCTCGTCCCGCGCTGGCCTTCAGTTCCCGGTAGGGCGAGTGCATCGCTTGCTGCGCAAAGGCAACTACGCGGAGCGAGTGGGGGCCGGCGCGCCCGTCTACATGGCTGCGGTCCTCGAGTATCTGACCGCCGAGATCCTGGAGCTGGCGGGCAACGCGGCTCGGGACAACAAGAAGACGCGCATCATCCCTCGTCACCTCCAGCTGGCCATCCGCAACGACGAGGAACTGAACAAGCTGCTGGGCAAAGTCACCATCGCCCAGGGCGGCGTCTTGCCTAACATCCAGGCCGTACTGCTCCCTAAGAAGACGGAGAGTCACCACAAGGCAAAGGGCAAG 57 H2B ATGCCTGAGCCAGCGAAATCCGCTCCCGCCCCGAAGAAGGGCTCCAAGAAGGCCGTGACCAAGGCGCAGAAGAAGGACGGCAAGAAGCGCAAGCGCAGCCGCAAGGAGAGCTACTCCGTATACGTGTACAAGGTGCTGAAACAGGTCCACCCCGACACCGGCATCTCCTCTAAAGCCATGGGGATCATGAATTCCTTTGTCAACGACATCTTCGAGCGCATCGCCGGCGAGGCTTCCCGCCTGGCGCATTACAACAAGCGCTCGACCATCACCTCCAGGGAGATCCAGACGGCCGTGCGCCTGCTGCTTCCCGGGGAGCTGGCCAAGCACGCTGTGTCAGAGGGCACCAAGGCCGTTACCAAGTACACCAGCTCCAAG 58 H4 ATGTCCGGCAGAGGAAAGGGCGGAAAAGGCTTAGGCAAAGGGGGCGCTAAGCGCCACCGCAAGGTCTTGAGAGACAACATTCAGGGCATCACCAAGCCTGCCATTCGGCGTCTAGCTCGGCGTGGCGGCGTTAAGCGGATCTCTGGCCTCATTTACGAGGAGACCCGCGGTGTGCTGAAGGTGTTCCTGGAGAATGTGATTCGGGACGCAGTCACCTACACCGAGCACGCCAAGCGCAAGACCGTCACAGCCATGGATGTGGTGTACGCGCTCAAGCGCCAGGGGCGCACCCTGTACGGCTTCGGAGGC 59 L1 GATCCACCGGTCGCCACC 60 NZ GCACTGAAAAAGGAACTGCAGGCTAACAAAAAGGAACTGGCACAGCTGAAGTGGGAGCTGCAGGCCCTGAAGAAAGAACTGGCTCAG 61 CZ GAGCAGCTGGAAAAGAAACTGCAGGCACTGGAGAAGAAACTGGCCCAGCTGGAATGGAAAAACCAGGCTCTGGAAAAGAAACTGGCACAG 62 Jun ATCGCAAGACTGGAGGAGAAGGTGAAGACACTGAAGGCACAGAATAGTGAGCTGGCCTCAACTGCTAACATGCTGCGAGAACAGGTGGCTCAGCTG 63 Fos CTGAAGGAGAAAGAAAAGCTGCTGAATGCAATCGAGACCCAGCTGGCCAGTAAAGAGGACGAACTGCAGGATACAGAAGCACAGCTGACCGATACA 64 L2-1 TCAGGGGGCACCTCTGGGTCAACATCTGGCACTGGCTCTACC 65 L2-2 AGTGGAGGGACCAGCGGATCCACCTCTGGAACAGGCTCCACT 66 L2-3 AGCGGCGGGACAAGCGGGAGCACCTCAGGGACTGGGAGCACT 67 L2-4 TCCGGCGGGACAAGCGGCAGCACATCCGGGACTGGCTCAACA 68 L2-5 AGCGGAGGGACCAGCGGGTCCACCTCTGGAACAGGCTCCACT 69 L2-6 TCTGGGGGCACCTCTGGCTCAACCTCTGGGACAGGCTCCACT

order
number denomination sequence list note 70 H3.1 MARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSSAVMALQEACEAYLVGLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERA 71 H3.2 MARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSSAVMALQEASEAYLVGLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERA 72 H3.3 MARTKQTARKSTGGKAPRKQLATKAARKSAPSTGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSAAIGALQEASEAYLVGLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERA 73 H2A.1 MSGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGNYAERVGAGAPVYLAAVLEYLTAEILELAGNAARDNKKTRIIPRHLQLAIRNDEELNKLLGKVTIAQGGVLPNIQAVLLPKKTESHHKAKGK 74 H2A.2 MSGRGKQGGKARAKAKSRSSRAGLQFPVGRVHRLLRKGNYAERVGAGAPVYMAAVLEYLTAEILELAGNAARDNKKTRIIPRHLQLAIRNDEELNKLLGKVTIAQGGVLPNIQAVLLPKKTESHHKAKGK 75 H2B MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPGELAKHAVSEGTKAVTKYTSSK 76 H4 MSGRGKGGKGLGKGGAKRHRKVLRDNIQGITKPAIRRLARRGGVKRISGLIYEETRGVLKVFLENVIRDAVTYTEHAKRKTVTAMDVVYALKRQGRTLYGFGG 77 L1 DPPVAT 78 NZ ALKKELQANKKELAQLKWELQALKKELAQ 79 CZ EQLEKKLQALEKKLAQLEWKNQALEKKLAQ 80 Jun IARLEEKVKTLKAQNSELASTANMLREQVAQL 81 Fos LKEKEKLLNAIETQLASKEDELQDTEAQLTDT 82 L2 SGGTSGSTSGTGST

HEK 293T cells were transformed using wild-type and mutant vectors having a genome containing the eGFP gene. Next, the transduction efficiency of wild-type and mutant vectors was quantified by counting cells expressing eGFP by flow cytometry, and the results are shown in FIG. 2 and Table 9.

Vectors Transducing units/mL WT 5.08E+06 37-H3.1 3.80E+04 37-H3.2 3.40E+04 37-H3.3 1.88E+04 273-H3.1 2.45E+05 273-H3.2 3.20E+05 273-H3.3 6.81E+05 273-H2A.1 4.46E+05 273-H2A.2 2.38E+05 273-H2B 7.90E+05 273-H4 3.67E+05 37-NZCZ 6.96E+04 37-JunFos 1.92E+05 273-NZCZ 1.30E+06 273-JunFos 1.22E+06

As can be seen in Figure 2 and Table 9, even when a foreign protein (107 to 154 amino acids in size) is introduced into the integrase, the mutant vectors showed a significant level of transduction ability into HEK 293T cells. For example, a sample of mutant 273-NZ-CZ exhibited a transducing titer of 1.3E + 06 T.U./mL. This titer level is 3.9-fold lower than that of the wild-type vector (5.1E + 06 T.U./mL). Overall, the transduction titer (2.4E + 05 TU/mL to 1.3E + 06 TU/mL) of the mutant in which the protein was inserted at the back of the integrase was higher than that of the mutant inserted at the front (1.9 E + 04 TU/mL to 1.9E + 05 TU/mL).

Frequency of vector gene insertions near the TSS region of the human genome

To map the gene insertion site of the retroviral vector, genomic DNA was isolated from HEK 293T cells transduced with wild-type and mutant vectors. The virus-cell genome junction was amplified by PCR and then analyzed by NGS method. Based on the analysis, 2,117 retroviral genome integration sites on the human genome were identified and the results are shown in Table 10.

Numbers of integration sites WT MLV 234 37-H3.1 268 37-H3.2 257 37-H3.3 126 273-H3.1 107 273-H3.2 102 273-H3.3 209 273-H2A.1 103 273-H2A.2 61 273-H2B 83 273-H4 94 37-NZ-CZ 77 37-Jun-Fos 111 273-NZ-CZ 117 273-Jun-Fos 168 Total 2,117

We also generated 1,000,000 computationally generated random sites in the human genome using the QuickMap tool (Appelt et al., 2009). Similar to the results of previous academic papers (Hacein-Bey-Abina et al., 2008; Deichmann et al., 2007; Howe et al., 2008; LaFave et al., 2014), wild-type MLV vectors have high genes in the TSS region. insertion frequency (TSS region is defined as a genomic site within 5 kb from the TSS of a protein-coding gene).

As can be seen in Fig. 3, the gene insertion frequency of wild-type viral vector into TSS was 42.3%, which was 6.8 times higher than the random probability (6.2%). The strong insertional preference of wild-type virus for the TSS region may result in the expression of oncogenes. The reason is that when the vector insertion site is adjacent to the TSS of the oncogene, the promoter(s) in the vector genome can activate that oncogene. Therefore, reducing vector insertion around the TSS region is very important for improving the safety of retroviral-based vectors.

In addition, as can be seen in FIG. 3 , a mutant equipped with a histone or leucine zipper is inserted into the TSS region at a frequency of 6.6-19.6%, and the frequency is significantly lower than that of the wild type. In particular, mutant 273-H2A.2 showed the lowest insertion frequency (6.6%) into the TSS region, which was 6.4-fold lower than that of the wild-type vector. The other two mutant vectors also showed significantly lower levels of insertion for the TSS region [273-H3.3 (7.7%) and 37-H3.1 (9.3%)].

When the retroviral vector is inserted into a genomic site closer to the TSS, the probability of activation of the gene downstream of the TSS will be higher. To confirm the possibility of this gene regulation perturbation, the spatial distribution of retroviral vector insertions around the TSS was further investigated. For this analysis, the genomic region containing the retroviral DNA was divided into smaller portions with a spatial interval of 1 kb starting at the TSS, as shown in FIGS. 4A and 4B.

As can be seen in Fig. 4a, the insertion of wild-type MLV was strongly enriched within 3 kb from TSS, whereas the mutant MLV did not show this concentrated insertion pattern. Looking at the larger extent around the TSS (50 kb in size), it is more clearly confirmed that the wild-type viral vector insertion is concentrated around the region closest to the TSS. In contrast, as can be seen in Fig. 4b, the mutant viral vector did not show a clear insertion pattern locally concentrated around the TSS.

<1kb <2kb <3kb <4kb <5kb Random 1.3% 2.5% 3.8% 5.0% 6.2% WT 19.7% 32.9% 40.2% 41.9% 42.3% 37-H3.1 2.6% 3.7% 6.0% 7.1% 9.3% 273-H3.3 2.4% 3.8% 4.5% 7.5% 8.2% 273-H2A.2 4.9% 6.6% 6.6% 6.6% 6.6%

In addition, as can be seen in Figure 4c and Table 11, the plot of cumulative retroviral insertion frequency as a function of distance from TSS clearly shows that insertion of wild-type vector into the human genome occurred at a very high frequency near the region within 1 kb of TSS. show Beyond the TSS-proximal region, the cumulative frequency of wild-type vector insertions did not increase significantly with distance from the TSS. This can be characterized as a saturation curve. In contrast, the cumulative insertion frequency of the two mutants (37-H3.1 and 273-H3.3) continued to increase with distance from the TSS, a pattern similar to that of random insertion. From this, it can be concluded that the insertion pattern of the mutant into the human genome is close to the random insertion pattern, which is very different from that of the wild type. In summary, the experimentally obtained vector insertion pattern results show that insertion of histone or leucine zippers into the integrase can effectively reduce the concentration of retroviral vector insertion around the TSS.

Frequency of retroviral insertion near carcinogenic sites

MLV vectors have shown cancer-causing potential, which is related to their high frequency insertion of the genome into the human genome near the oncogene TSS. In past gene therapy clinical cases, the MLV vector was inserted into the LMO2 (LIM domain only 2) enhancer region, one of the oncogenes. This has resulted in leukemia in young patients. In order to compare the carcinogenic potential of the mutant vector with that of the wild-type vector, the frequency of vector insertion into a genomic site within 5 kb from the oncogene TSS was quantified using the QuickMap tool, and the results are shown in Figures 5 and 12 and Table 13. indicated.

Number of integrations Number of integrations within 5 kb of TSSs Frequency of integrations within 5 kb of TSSs (%) P-value (compared with the frequency
of wild-type) Random 1,000,000 61,617 6.2** 2.98E-56 WT 234 99 42.3 37-H3.1 268 25 9.3** 3.45E-18 37-H3.2 257 25 9.7** 2.72E-17 37-H3.3 126 22 17.5** 8.80E-07 273-H3.1 107 13 12.1** 7.64E-09 273-H3.2 102 20 19.6** 3.42E-05 273-H3.3 209 16 7.7** 5.58E-18 273-H2A.1 103 12 11.7** 6.39E-09 273-H2A.2 61 4 6.6** 1.40E-08 273-H2B 83 9 10.8** 3.61E-08 273-H4 94 16 17.0** 6.39E-06 37-NZ-CZ 77 12 15.6** 8.98E-06 37-Jun-Fos 111 17 15.3** 2.54E-07 273-NZ-CZ 117 21 17.9** 2.73E-06 273-Jun-Fos 168 28 16.7** 2.22E-08

If the test P-value for the difference compared to that of the wild-type vector is less than 0.05, the insertion frequency is indicated by **.

Number of integrations Number of integrations near the TSSs of
oncogenes Frequency of integrations near the TSSs of oncogenes (%) P-value (compared with the frequency of wild-type) Random 1,000,000 1,577 0.16** 4.08E-05 WT 234 5 2.14 37-H3.1 268 One 0.37* 7.95E-02 37-H3.2 257 0 0.00** 2.40E-02 37-H3.3 126 2 1.59 5.32E-01 273-H3.1 107 One 0.93 3.89E-01 273-H3.2 102 One 0.98 4.11E-01 273-H3.3 209 2 0.96 2.74E-01 273-H2A.1 103 0 0.00 1.59E-01 273-H2A.2 61 0 0.00 3.11E-01 273-H2B 83 0 0.00 2.17E-01 273-H4 94 0 0.00 1.83E-01 37-NZ-CZ 77 0 0.00 2.39E-01 37-Jun-Fos 111 0 0.00 1.42E-01 273-NZ-CZ 117 0 0.00 1.30E-01 273-Jun-Fos 168 0 0.00* 6.56E-02

If the test P-values for differences compared to that of the wild-type vector were less than 0.1 and 0.05, respectively, insertion frequencies are denoted by * and **

As can be seen in Figure 5 and Table 12, the frequency of wild-type MLV insertions into these genomic regions (2.14%) was 13.4 times higher than that in the random case (0.16%). Wild-type vectors were inserted near five oncogenes TSS: PCM1, HOXD11, RB1, GATA2 and NACA.

On the other hand, as can be seen in Figure 5 and Table 13, the insertion frequency of the three mutants (37-H3.1, 37-H3.2 and 273-Jun-Fos) for these regions was much lower than that of the wild type (P- value < 0.1). Mutants 37-H3.1 and 273-H3.1 were inserted near the TSS of only one oncogene PBRM1 (Table 12). 273-H3.2 was inserted near the TSS of the EVI1 oncogene and another mutant 37-H3.3 was inserted near the TSS of two oncogenes, PBRM1 and TLX3. Mutant 273-H3.3 was independently inserted twice near the TSS of the oncogene ERG. It is noteworthy that mutant 37-H3.2 did not insert into the oncogenic region (P-value < 0.05). These results show that inserting histone or leucine zippers inside the integrase can significantly change the retroviral vector insertion pattern, thereby significantly reducing the carcinogenic potential. No other mutants were inserted into the oncogenic region, but the number of virus-cell genome conjugate reads was not sufficient to obtain a statistically significant P-value.

Retroviral vector insertion near CpG islands and cis-regulatory elements

CpG islands and cis-regulatory elements are often located near the promoter region of a gene. Thus, retroviral insertion into genomic regions near CpG islands and cis-regulatory elements can also lead to perturbation of gene expression regulation. Wild-type MLV highly favored CpG islands and cis-regulators during insertion.

Number of integrations Number of integrations into the oncogenic locations Frequency of integrations into the oncogenic locations (%) Oncogenes whose TSSs were located near retroviral integration sites (within 5 kb) WT 234 5 2.14 PCM1
HOXD11
RB1
GATA2
NACA 37-H3.1 268 One 0.37 PBRM1 37-H3.3 126 2 1.59 PBRM1TLX3 273-H3.1 107 One 0.93 PBRM1 273-H3.2 102 One 0.98 EVI1 273-H3.3 209 2 0.96 ERG (two integrations)

Number of integrations Number of integrations
within 5 kb of
CpG islands Frequency of
integrations within 5
kb of CpG islands
(%) P-value (compared
with the frequency
of wild-type) Random 1,000,000 71,763 7.2** 2.33E-49 WT 234 98 41.9 37-H3.1 268 21 7.8** 6.09E-20 37-H3.2 257 15 5.8** 1.11E-22 37-H3.3 126 28 22.2** 1.17E-04 273-H3.1 107 19 17.8** 6.38E-06 273-H3.2 102 22 21.6** 2.11E-04 273-H3.3 209 17 8.1** 4.66E-17 273-H2A.1 103 14 13.6** 1.09E-07 273-H2A.2 61 10 16.4** 1.16E-04 273-H2B 83 16 19.3** 1.25E-04 273-H4 94 18 19.1** 5.30E-05 37-NZ-CZ 77 14 18.2** 8.97E-05 37-Jun-Fos 111 23 20.7** 6.88E-05 273-NZ-CZ 117 24 20.5** 4.22E-05 273-Jun-Fos 168 22 13.1** 1.36E-10

If the test P-value for the difference compared to that of the wild-type vector is less than 0.05, the insertion frequency is indicated by **.

Number of integrations Number of integrations within 5 kb of cis-regulatory elements Frequency of integrations within 5 kb of cis-regulatory
elements (%) P-value (compared with the frequency of wild-type) Random 1,000,000 68,572 6.86** 4.05E-55 WT 234 102 43.6 37-H3.1 268 24 9.0** 9.00E-20 37-H3.2 257 29 11.3** 2.14E-16 37-H3.3 126 30 23.8** 1.27E-04 273-H3.1 107 16 15.0** 7.86E-08 273-H3.2 102 21 20.6** 3.16E-05 273-H3.3 209 18 8.6** 9.90E-18 273-H2A.1 103 15 14.6** 7.55E-08 273-H2A.2 61 9 14.8** 1.40E-05 273-H2B 83 8 9.6** 2.71E-09 273-H4 94 14 14.9** 2.89E-07 37-NZ-CZ 77 13 16.9** 1.14E-05 37-Jun-Fos 111 12 10.8** 1.84E-10 273-NZ-CZ 117 23 19.7** 5.27E-06 273-Jun-Fos 168 27 16.1** 2.19E-09

If the test P-value for the difference compared to that of the wild-type vector is less than 0.05, the insertion frequency is indicated by **.

6A and 6B , wild-type MLV insertions mainly occurred at genomic sites within 5 kb from CpG islands (41.9% frequency) and cis-regulatory elements (43.6% frequency). In contrast, integrase mutations significantly reduced the insertion preference for these regions. Mutant vectors were inserted within 5 kb of CpG islands at frequencies of 5.8-22.2% and within 5 kb of cis-regulatory elements at frequencies of 8.6-23.8%. In addition, the CpG island region insertion frequencies of the three mutants selected as safe mutation vectors based on the low insertion frequency into the TSS region were 7.7% (37-H3.1), 8.1% (273-H3.3)) and 16.4 % (273-H2A.2). These frequencies are 5.4-fold, 5.2-fold and 2.6-fold lower than that of the wild-type vector.

On the other hand, as can be seen in Figure 6b, the insertion of histone inside the integrase made the insertion of the mutant vector away from the cis-regulatory element. The insertion frequency of mutant 273-H3.3 to a genomic site within 5 kb of the cis-regulatory element was 8.6%, 5.1-fold lower than that of the wild-type vector. In addition, mutants 37-H3.1 and 273-H2A.2 were inserted near the cis-regulatory domain at frequencies of 8.9% and 9.8% (4.9-fold and 4.5-fold lower) than wild-type, respectively.

In addition, the retroviral vector insertion site within 5 kb of the CpG island and the cis-regulatory element position was divided into subsets with an interval of 1 kb starting from the corresponding genomic component, and the results are shown in FIG. 7A .

As can be seen in Figure 7a, wild-type MLV insertion was concentrated within 2 kb from the CpG island. In contrast, the two mutant vectors 37-H3.1 and 273-H3.3 did not show strong intensive insertion near CpG islands.

As can be seen in FIG. 7B , the concentration of wild-type vector insertion near the CpG island can be more clearly identified when looking at a larger area around the CpG island (within a region of 50 kb from the CpG island).

< 1 kb < 2 kb < 3 kb < 4 kb < 5 kb Random 1.6% 3.1% 4.5% 5.8% 7.2% WT 23.1% 37.2% 39.7% 40.6% 41.9% 37-H3.1 1.5% 3.4% 5.2% 6.0% 7.8% 273-H3.3 2.4% 3.8% 4.1% 6.3% 7.1% 273-H2A.2 11.5% 14.8% 14.8% 16.4% 16.4%

As can be seen in Figure 7c and Table 17, analysis of the cumulative frequency of retroviral vector insertions according to distance from CpG islands shows that wild-type vector insertions are highly concentrated around CpG islands. On the other hand, mutants 37-H3.1 and 273-H3.3 did not show a highly concentrated insertion pattern around CpG islands. Rather, the mutants show a linear increase in insertion frequency with distance from the CpG island, similar to the random case. More quantitatively, 37.2% of wild-type MLV insertions occurred within 2 kb of CpG islands. However, in the case of 37-H3.2 and 273-H3.3, insertions occurred in the corresponding regions in only 3.4% and 3.8% of cases. Mutant 273-H2A.2 was inserted into the same genomic region with a higher frequency (14.8%) than the other mutants. However, this frequency is still 2.5 times lower than for wild-type vectors.

< 1 kb < 2 kb < 3 kb < 4 kb < 5 kb Random 2.2% 3.4% 4.6% 5.7% 6.9% WT 29.9% 36.8% 40.6% 42.7% 43.6% 37-H3.1 3.7% 4.9% 6.0% 7.8% 9.0% 273-H3.3 3.3% 3.3% 5.6% 7.1% 8.2% 273-H2A.2 6.6% 6.6% 8.2% 9.8% 14.8%

As can be seen in Figure 7d and Table 18, similarly, wild-type MLV human genome insertions were highly concentrated within 2 kb from cis-regulatory elements (frequency: 36.8%), whereas mutants were highly concentrated near cis-regulatory elements. It did not show a concentrated insertion tendency. For example, insertion of mutant 273-H3.3 occurred at a frequency of 3.3%, 11.2 fold lower than for wildtype, within 2 kb from the cis-regulatory element. Mutant vectors 37-H3.1 and 273-H3.3 also exhibited a form of cumulative frequency similar to that of random insertion (increasing linearly with distance from the cis-regulatory element). From all the obtained results, it was concluded that by inserting a histone or leucine zipper inside the MLV integrase, it is possible to substantially reduce the intrinsic insertion preference of retroviruses for the human genome regulatory region.

Structure prediction of mutant integrase

Structural perturbation of integrase by internal introduction of DNA-binding proteins would be expected to shift MLV insertions away from regions regulating expression on the human genome. Since the structure of wild-type MLV integrase has not yet been elucidated, the structure of the obtained mutant integrase is also difficult to experimentally elucidate. Therefore, the present inventors used I-TASSER (Zhang, 2008), which is one of the most advanced protein structure prediction tools, instead of trying to unravel the structure of the mutant integrase experimentally. It was predicted theoretically.

MLV integrase is known to contain a number of structural subdomains, an N-terminal extension domain (NED), an N-terminal domain (NTD), a catalytic core domain (CCD) and a C-terminal domain (CTD).

As can be seen in FIG. 8a , when a histone is inserted at the 273 th amino acid residue position of integrase, a change in the structure of the CTD is induced as shown in the example of 273-H3.3. To understand the potential reasons for this structural perturbation, we analyzed the newly formed hydrogen bonds between the 'integrase sub-d main' and the 'histone inserted into the mutant integrase'.

As can be seen in Figure 8b, 13 new hydrogen bonds occurred between histones and three integrase subdomains (CCD, linker and CTD; Figure 8B). The inserted H3.3 histone is hydrogen-bonded with two amino acids of CCD (286K (lower, integrase partial amino acids indicated by "i"))-437T (lower, histone partial amino acids indicated by "h") and 286K (i) )-438R(h); 8b) is formed. The inserted H3.3 histone is an integrase linker and 7 hydrogen bonds (285T(i)-442S(h), 287Q(i)-442S(h), 405D(h)-473R(i), 409A(h) )-473R(i), 413R(h)-472D(i), 413R(h)-473R(i), 421V(h)-470Q(i)).

As can be seen in Fig. 8a, this new hydrogen bonding may in part cause disturbance of the CCD and linker structures. In addition, as can be seen in FIG. 8b, there are four hydrogen bonds between H3.3 and CTD (351R(h)-495N(i), 387E(h)-489R(i), 387E(h)-527H(i)) , 390N(h)-529K(i)) were present. This novel interaction could lead to partial disruption of the beta sheet in CTD.

order
number denomination Sequence Listing (5'-> 3') note 83 Integrases_DNA sequence atagaaaattcatcaccctacacctcagaacattttcattacacagtgactgatataaaggacctaaccaagttgggggccatttatgataaaacaaagaagtattgggtctaccaaggaaaacctgtgatgcctgaccagtttacttttgaattattagactttcttcatcagctgactcacctcagcttctcaaaaatgaaggctctcctagagagaagccacagtccctactacatgctgaaccgggatcgaacactcaaaaatatcactgagacctgcaaagcttgtgcacaagtcaacgccagcaagtctgccgttaaacagggaactagggtccgcgggcatcggcccggcactcattgggagatcgatttcaccgagataaagcccggattgtatggctataaatatcttctagtttttatagataccttttctggctggatagaagccttcccaaccaagaaagaaaccgccaaggtcgtaaccaagaagctactagaggagatcttccccaggttcggcatgcctcaggtattgggaactgacaatgggcctgccttcgtctccaaggtgagtcagacagtggccgatctgttggggattgattggaaattacattgtgcatacagaccccaaagctcaggccaggtagaaagaatgaatagaaccatcaaggagactttaactaaattaacgcttgcaactggctctagagactgggtgctcctactccccttagccctgtaccgagcccgcaacacgccgggcccccatggcctcaccccatatgagatcttatatggggcacccccgccccttgtaaacttccctgaccctgacatgacaagagttactaacagcccctctctccaagctcacttacaggctctctacttagtccagcacgaagtctggagacctctggcggcagcctaccaagaacaactggaccgaccggtggtacctcacccttaccgagtcggcgacacagtgt gggtccgccgacaccagactaagaacctagaacctcgctggaaaggaccttacacagtcctgctgaccacccccaccgccctcaaagtagacggcatcgcagcttggatacacgccgcccacgtgaaggctgccgaccccggtaccgtggaccatcctctagactgacacatggctagcgttacca

order
number denomination sequence list note 84 Integrases_Amino acid sequence iensspytsehfhytvtdikdltklgaiydktkkywvyqgkpvmpdqftfelldflhqlthlsfskmkallershspyymlnrdrtlknitetckacaqvnasksavkqgtrvrghrpgthweidfteikpglygykyllvfidtfsgwieafptkketakvvtkklleeifprfgmpqvlgtdngpafvskvsqtvadllgidwklhcayrpqssgqvermnrtiketltkltlatgsrdwvlllplalyrarntpgphgltpyeilygappplvnfpdpdmtrvtnspslqahlqalylvqhevwrplaaayqeqldrpvvphpyrvgdtvwvrrhqtknleprwkgpytvllttptalkvdgiaawihaahvkaadpgggpssrltwrvqrsqnplkirltreap

<110> Sookmyung Women's university industry-academic cooperation foundation <120> Safe retroviral vectors developed by insertion of histone or leucine zipper into the viral integrase <130> PN200017 <160> 82 <170> KoPatentIn 3.0 <210> 1 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 1 Asp Asp Pro Val Ala Thr 1 5 <210> 2 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> H2A_sense_primer <400> 2 aaaaagcgct gatccaccgg tcgccaccat gtctggacgt ggcaagcag 49 <210> 3 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> H2A_antisense_primer <400> 3 aaaagctagc ggtggcgacc ggtggatcct tgcccttagc tttgtggtgg 50 <210> 4 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> H2B_sense_primer <400> 4 aaaaggcgcc gatccaccgg tcgccaccat gcctgagcca gcgaaatcc 49 <210> 5 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> H2B_antisense_primer <400> 5 aaaagctagc ggtggcgacc ggtggatcct tggagctggt gtacttggta acg 53 <210> 6 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> H3.1_sense_primer <400> 6 aaaaagcgct gatccaccgg tcgccaccat ggcacgcacg aagcaaac 48 <210> 7 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> H3.1_antisense_primer <400> 7 aaaagctagc ggtggcgacc ggtggatctg ccctctctcc gcgaatg 47 <210> 8 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> H3.2_sense_primer <400> 8 aaaaagcgct gatccaccgg tcgccaccat ggcccgtact aagcagactg c 51 <210> 9 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> H3.2_antisense_primer <400> 9 aaaagctagc ggtggcgacc ggtggatcag cccgctctcc acggatg 47 <210> 10 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> H3.3_sense_primer <400> 10 aaaaagcgct gatccaccgg tcgccaccat ggctcgtaca aagcagactg cc 52 <210> 11 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> H3.3_antisense_primer <400> 11 aaaagctagc ggtggcgacc ggtggatcag cacgttctcc acgtatgcg 49 <210> 12 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> H4_sense_primer <400> 12 aaaaagcgct gatccaccgg tcgccaccat gtccggcaga ggaaaggg 48 <210> 13 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> H4_antisense_primer <400> 13 aaaaggcgcc gatccaccgg tcgccaccat gcctgagcca gcgaaatcc 49 <210> 14 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> biotinylated oligonucleotide <400> 14 atttgttaaa gavaggatat cagtggtcca g 31 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> linker+ <400> 15 gtaatacgac tcactatagg gctccgctta agggac 36 <210> 16 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Linker- <400> 16 tagtccctta gcggag 16 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer_F <400> 17 gacttgtggt ctcgctgttc cttgg 25 <210> 18 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primer_R <400> 18 gtaatacgac tcactatagg gctccccgct taag 34 <210> 19 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for adapter addition <400> 19 tcgtcggcag cgtcagatgt gtataagaga cagggagggt ctcctctgag tgattgacta 60 cc 62 <210> 20 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for adapter addition <400> 20 gtctcgtggg ctcggagatg tgtataagag acagactcac tatagggctc cgcttaaggg 60 ac 62 <210> 21 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for index addition <400> 21 aatgatacgg cgaccaccga gatctacact cgtcggcagc gtc 43 <210> 22 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for index addition <400> 22 caagcagaag acggcatacg agatrrrrrr rrgtctcgtg ggctcgg 47 <210> 23 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> FFFFFFFF_S502 <400> 23 ctctctat 8 <210> 24 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> FFFFFFFF_S503 <400> 24 tatcctct 8 <210> 25 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> FFFFFFFF_S505 <400> 25 gtaaggag 8 <210> 26 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> FFFFFFFF_S506 <400> 26 actgcata 8 <210> 27 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> FFFFFFFF_S507 <400> 27 aaggagta 8 <210> 28 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> FFFFFFFF_S508 <400> 28 ctaagcct 8 <210> 29 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> FFFFFFFF_S510 <400> 29 cgtctaat 8 <210> 30 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> FFFFFFFF_S511 <400> 30 tctctccg 8 <210> 31 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> FFFFFFFF_S513 <400> 31 tcgactag 8 <210> 32 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> FFFFFFFF_S515 <400> 32 ttctagct 8 <210> 33 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> FFFFFFFF_S516 <400> 33 cctaggt 8 <210> 34 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> FFFFFFFF_S517 <400> 34 gcgtaaga 8 <210> 35 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> FFFFFFFF_S518 <400> 35 ctattag 8 <210> 36 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> FFFFFFFF_S520 <400> 36 aaggctat 8 <210> 37 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> FFFFFFFF_S521 <400> 37 gagcctta 8 <210> 38 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> FFFFFFFF_S522 <400> 38 ttatgcga 8 <210> 39 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> RRRRRRRR_N701 <400> 39 tcgcctta 8 <210> 40 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> RRRRRRRR_N702 <400> 40 ctagtacg 8 <210> 41 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> RRRRRRRR_N703 <400> 41 ttctgcct 8 <210> 42 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> RRRRRRRR_N704 <400> 42 gctcagga 8 <210> 43 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> RRRRRRRR_N705 <400> 43 aggagtcc 8 <210> 44 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> RRRRRRRR_N706 <400> 44 catgccta 8 <210> 45 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> RRRRRRRR_N707 <400> 45 gtagagag 8 <210> 46 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> RRRRRRRR_N710 <400> 46 cagcctcg 8 <210> 47 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> RRRRRRRR_N711 <400> 47 tgcctctt 8 <210> 48 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> RRRRRRRR_N712 <400> 48 tcctctac 8 <210> 49 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> RRRRRRRR_N714 <400> 49 tcatgagc 8 <210> 50 <211> 8 <212> DNA <213> Artificial Sequence <220> <223> RRRRRRRR_N715 <400> 50 cctgagat 8 <210> 51 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> viral 3 LTR sequence <400> 51 ggagggtctc ctctgagtga ttgactaccc gtcagcgggg gtctttca 48 <210> 52 <211> 408 <212> DNA <213> Artificial Sequence <220> <223> H3.1 <400> 52 atggcacgca cgaagcaaac agctcgtaag tccactggcg gcaaagcccc gcgcaagcag 60 ctggccacta aggcggctcg caaaagcgcg ccagccaccg gtggcgtgaa gaagccccac 120 cgctacaggc ctggtactgt cgccctccgt gaaatccgcc gctatcagaa atcgactgag 180 ctactgattc gcaagctacc attccagcgt ctggtacgtg agatcgcgca ggacttcaag 240 accgacctgc gcttccagag ctcggctgtg atggcgctgc aggaggcctg cgaggcttac 300 ctggtggggc tctttgagga caccaacctg tgtgctatcc acgccaagcg agtgactatc 360 atgcccaagg acatccagct cgctcgccgc attcgcggag agagggca 408 <210> 53 <211> 408 <212> DNA <213> Artificial Sequence <220> <223> H3.2 <400> 53 atggcccgta ctaagcagac tgctcgcaag tcgaccggcg gcaaggcccc gaggaagcag 60 ctggccacca aggcggcccg caagagcgcg ccggccacgg gcggggtgaa gaagccgcac 120 cgctaccggc ccggcaccgt agccctgcgg gagatccggc gctaccagaa gtccacggag 180 ctgctgatcc gcaagctgcc cttccagcgg ctggtacgcg agatcgcgca ggactttaag 240 acggacctgc gcttccagag ctcggccgtg atggcgctgc aggaggccag cgaggcctac 300 ctggtggggc tgttcgaaga cacgaacctg tgcgccatcc acgccaagcg cgtgaccatt 360 atgcccaagg acatccagct ggcccgccgc atccgtggag agcgggct 408 <210> 54 <211> 408 <212> DNA <213> Artificial Sequence <220> <223> H3.3 <400> 54 atggctcgta caaagcagac tgcccgcaaa tcgaccggtg gtaaagcacc caggaagcaa 60 ctggctacaa aagccgctcg caagagtgcg ccctctaccg gaggggtgaa gaaacctcat 120 cgttacaggc ctggtactgt ggcgctccgt gaaattagac gttatcagaa gtccactgaa 180 cttctgattc gcaaacttcc cttccagcgt ctggtgcgag aaattgctca ggactttaaa 240 acagatctgc gcttccagag cgcagctatc ggtgctttgc aggaggcaag tgaggcctat 300 ctggttggcc tttttgaaga caccaacctg tgtgctatcc atgccaaacg tgtaacaatt 360 atgccaaaag acatccagct ggcacgccgc atacgtggag aacgtgct 408 <210> 55 <211> 390 <212> DNA <213> Artificial Sequence <220> <223> H2A.1 <400> 55 atgtctggac gtggcaagca gggcggcaag gctcgcgcca aggccaaaac ccgctcctct 60 agagctgggc tccaatttcc tgtaggacga gtgcaccgcc tgctccgcaa gggcaactac 120 gctgagcggg tcggggccgg cgcgccggtt tacctggcgg cggtgctgga gtacctaact 180 gccgagatcc tggagctggc gggcaacgca gcccgcgaca acaaaaagac ccgcatcatc 240 ccgcgccact tgcagctggc catccgcaac gacgaggagc tcaacaagct gcttggtaaa 300 gttaccatcg ctcagggcgg tgttctgcct aacatccagg ccgtactgct ccccaagaag 360 actgagagcc accacaaagc taagggcaag 390 <210> 56 <211> 390 <212> DNA <213> Artificial Sequence <220> <223> H2A.2 <400> 56 atgtctggtc gtggcaagca aggaggcaag gcccgcgcca aggccaagtc gcgctcgtcc 60 cgcgctggcc ttcagttccc ggtagggcga gtgcatcgct tgctgcgcaa aggcaactac 120 gcggagcgag tgggggccgg cgcgcccgtc tacatggctg cggtcctcga gtatctgacc 180 gccgagatcc tggagctggc gggcaacgcg gctcgggaca acaagaagac gcgcatcatc 240 cctcgtcacc tccagctggc catccgcaac gacgaggaac tgaacaagct gctgggcaaa 300 gtcaccatcg cccagggcgg cgtcttgcct aacatccagg ccgtactgct ccctaagaag 360 acggagagtc accacaaggc aaagggcaag 390 <210> 57 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> H2B <400> 57 atgcctgagc cagcgaaatc cgctcccgcc ccgaagaagg gctccaagaa ggccgtgacc 60 aaggcgcaga agaaggacgg caagaagcgc aagcgcagcc gcaaggagag ctactccgta 120 tacgtgtaca aggtgctgaa acaggtccac cccgacaccg gcatctcctc taaagccatg 180 gggatcatga attcctttgt caacgacatc ttcgagcgca tcgccggcga ggcttcccgc 240 ctggcgcatt acaacaagcg ctcgaccatc acctccaggg agatccagac ggccgtgcgc 300 ctgctgcttc ccggggagct ggccaagcac gctgtgtcag agggcaccaa ggccgttacc 360 aagtacacca gctccaag 378 <210> 58 <211> 309 <212> DNA <213> Artificial Sequence <220> <223> H4 <400> 58 atgtccggca gaggaaaggg cggaaaaggc ttaggcaaag ggggcgctaa gcgccaccgc 60 aaggtcttga gagacaacat tcagggcatc accaagcctg ccattcggcg tctagctcgg 120 cgtggcggcg ttaagcggat ctctggcctc atttacgagg agacccgcgg tgtgctgaag 180 gtgttcctgg agaatgtgat tcgggacgca gtcacctaca ccgagcacgc caagcgcaag 240 accgtcacag ccatggatgt ggtgtacgcg ctcaagcgcc aggggcgcac cctgtacggc 300 ttcggaggc 309 <210> 59 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> L1 <400> 59 gatccaccgg tcgccacc 18 <210> 60 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> NZ <400> 60 gcactgaaaa aggaactgca ggctaacaaa aaggaactgg cacagctgaa gtgggagctg 60 caggccctga agaaagaact ggctcag 87 <210> 61 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> CZ <400> 61 gagcagctgg aaaagaaact gcaggcactg gagaagaaac tggcccagct ggaatggaaa 60 aaccaggctc tggaaaagaa actggcacag 90 <210> 62 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> Jun <400> 62 atcgcaagac tggaggagaa ggtgaagaca ctgaaggcac agaatagtga gctggcctca 60 actgctaaca tgctgcgaga acaggtggct cagctg 96 <210> 63 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> <400> 63 ctgaaggaga aagaaaagct gctgaatgca atcgagaccc agctggccag taaagaggac 60 gaactgcagg atacagaagc acagctgacc gataca 96 <210> 64 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> L2-1 <400> 64 tcagggggca cctctgggtc aacatctggc actggctcta cc 42 <210> 65 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> L2-2 <400> 65 agtggaggga ccagcggatc cacctctgga acaggctcca ct 42 <210> 66 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> L2-3 <400> 66 agcggcggga caagcgggag cacctcaggg actgggagca ct 42 <210> 67 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> L2-4 <400> 67 tccggcggga caagcggcag cacatccggg actggctcaa ca 42 <210> 68 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> L2-5 <400> 68 agcggaggga ccagcgggtc cacctctgga acaggctcca ct 42 <210> 69 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> L2-6 <400> 69 tctgggggca cctctggctc aacctctggg acaggctcca ct 42 <210> 70 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> H3.1 <400> 70 Met Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala 1 5 10 15 Pro Arg Lys Gln Leu Ala Thr Lys Ala Ala Arg Lys Ser Ala Pro Ala 20 25 30 Thr Gly Gly Val Lys Lys Pro His Arg Tyr Arg Pro Gly Thr Val Ala 35 40 45 Leu Arg Glu Ile Arg Arg Tyr Gln Lys Ser Thr Glu Leu Leu Ile Arg 50 55 60 Lys Leu Pro Phe Gln Arg Leu Val Arg Glu Ile Ala Gln Asp Phe Lys 65 70 75 80 Thr Asp Leu Arg Phe Gln Ser Ser Ala Val Met Ala Leu Gln Glu Ala 85 90 95 Cys Glu Ala Tyr Leu Val Gly Leu Phe Glu Asp Thr Asn Leu Cys Ala 100 105 110 Ile His Ala Lys Arg Val Thr Ile Met Pro Lys Asp Ile Gln Leu Ala 115 120 125 Arg Arg Ile Arg Gly Glu Arg Ala 130 135 <210> 71 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> H3.2 <400> 71 Met Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala 1 5 10 15 Pro Arg Lys Gln Leu Ala Thr Lys Ala Ala Arg Lys Ser Ala Pro Ala 20 25 30 Thr Gly Gly Val Lys Lys Pro His Arg Tyr Arg Pro Gly Thr Val Ala 35 40 45 Leu Arg Glu Ile Arg Arg Tyr Gln Lys Ser Thr Glu Leu Leu Ile Arg 50 55 60 Lys Leu Pro Phe Gln Arg Leu Val Arg Glu Ile Ala Gln Asp Phe Lys 65 70 75 80 Thr Asp Leu Arg Phe Gln Ser Ser Ala Val Met Ala Leu Gln Glu Ala 85 90 95 Ser Glu Ala Tyr Leu Val Gly Leu Phe Glu Asp Thr Asn Leu Cys Ala 100 105 110 Ile His Ala Lys Arg Val Thr Ile Met Pro Lys Asp Ile Gln Leu Ala 115 120 125 Arg Arg Ile Arg Gly Glu Arg Ala 130 135 <210> 72 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> H3.3 <400> 72 Met Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala 1 5 10 15 Pro Arg Lys Gln Leu Ala Thr Lys Ala Ala Arg Lys Ser Ala Pro Ser 20 25 30 Thr Gly Gly Val Lys Lys Pro His Arg Tyr Arg Pro Gly Thr Val Ala 35 40 45 Leu Arg Glu Ile Arg Arg Tyr Gln Lys Ser Thr Glu Leu Leu Ile Arg 50 55 60 Lys Leu Pro Phe Gln Arg Leu Val Arg Glu Ile Ala Gln Asp Phe Lys 65 70 75 80 Thr Asp Leu Arg Phe Gln Ser Ala Ala Ile Gly Ala Leu Gln Glu Ala 85 90 95 Ser Glu Ala Tyr Leu Val Gly Leu Phe Glu Asp Thr Asn Leu Cys Ala 100 105 110 Ile His Ala Lys Arg Val Thr Ile Met Pro Lys Asp Ile Gln Leu Ala 115 120 125 Arg Arg Ile Arg Gly Glu Arg Ala 130 135 <210> 73 <211> 130 <212> PRT <213> Artificial Sequence <220> <223> H2A.1 <400> 73 Met Ser Gly Arg Gly Lys Gln Gly Gly Lys Ala Arg Ala Lys Ala Lys 1 5 10 15 Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His 20 25 30 Arg Leu Leu Arg Lys Gly Asn Tyr Ala Glu Arg Val Gly Ala Gly Ala 35 40 45 Pro Val Tyr Leu Ala Ala Val Leu Glu Tyr Leu Thr Ala Glu Ile Leu 50 55 60 Glu Leu Ala Gly Asn Ala Ala Arg Asp Asn Lys Lys Thr Arg Ile Ile 65 70 75 80 Pro Arg His Leu Gln Leu Ala Ile Arg Asn Asp Glu Glu Leu Asn Lys 85 90 95 Leu Leu Gly Lys Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile 100 105 110 Gln Ala Val Leu Leu Pro Lys Lys Thr Glu Ser His His Lys Ala Lys 115 120 125 Gly Lys 130 <210> 74 <211> 130 <212> PRT <213> Artificial Sequence <220> <223> H2A.2 <400> 74 Met Ser Gly Arg Gly Lys Gln Gly Gly Lys Ala Arg Ala Lys Ala Lys 1 5 10 15 Ser Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His 20 25 30 Arg Leu Leu Arg Lys Gly Asn Tyr Ala Glu Arg Val Gly Ala Gly Ala 35 40 45 Pro Val Tyr Met Ala Ala Val Leu Glu Tyr Leu Thr Ala Glu Ile Leu 50 55 60 Glu Leu Ala Gly Asn Ala Ala Arg Asp Asn Lys Lys Thr Arg Ile Ile 65 70 75 80 Pro Arg His Leu Gln Leu Ala Ile Arg Asn Asp Glu Glu Leu Asn Lys 85 90 95 Leu Leu Gly Lys Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile 100 105 110 Gln Ala Val Leu Leu Pro Lys Lys Thr Glu Ser His His Lys Ala Lys 115 120 125 Gly Lys 130 <210> 75 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> H2B <400> 75 Met Pro Glu Pro Ala Lys Ser Ala Pro Ala Pro Lys Lys Gly Ser Lys 1 5 10 15 Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Gly Lys Lys Arg Lys Arg 20 25 30 Ser Arg Lys Glu Ser Tyr Ser Val Tyr Val Tyr Lys Val Leu Lys Gln 35 40 45 Val His Pro Asp Thr Gly Ile Ser Ser Lys Ala Met Gly Ile Met Asn 50 55 60 Ser Phe Val Asn Asp Ile Phe Glu Arg Ile Ala Gly Glu Ala Ser Arg 65 70 75 80 Leu Ala His Tyr Asn Lys Arg Ser Thr Ile Thr Ser Arg Glu Ile Gln 85 90 95 Thr Ala Val Arg Leu Leu Leu Pro Gly Glu Leu Ala Lys His Ala Val 100 105 110 Ser Glu Gly Thr Lys Ala Val Thr Lys Tyr Thr Ser Ser Lys 115 120 125 <210> 76 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> H4 <400> 76 Met Ser Gly Arg Gly Lys Gly Gly Lys Gly Leu Gly Lys Gly Gly Ala 1 5 10 15 Lys Arg His Arg Lys Val Leu Arg Asp Asn Ile Gln Gly Ile Thr Lys 20 25 30 Pro Ala Ile Arg Arg Leu Ala Arg Arg Gly Gly Val Lys Arg Ile Ser 35 40 45 Gly Leu Ile Tyr Glu Glu Thr Arg Gly Val Leu Lys Val Phe Leu Glu 50 55 60 Asn Val Ile Arg Asp Ala Val Thr Tyr Thr Glu His Ala Lys Arg Lys 65 70 75 80 Thr Val Thr Ala Met Asp Val Val Tyr Ala Leu Lys Arg Gln Gly Arg 85 90 95 Thr Leu Tyr Gly Phe Gly Gly 100 <210> 77 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> L1 <400> 77 Asp Pro Pro Val Ala Thr 1 5 <210> 78 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> NZ <400> 78 Ala Leu Lys Lys Glu Leu Gln Ala Asn Lys Lys Glu Leu Ala Gln Leu 1 5 10 15 Lys Trp Glu Leu Gln Ala Leu Lys Lys Glu Leu Ala Gln 20 25 <210> 79 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> CZ <400> 79 Glu Gln Leu Glu Lys Lys Leu Gln Ala Leu Glu Lys Lys Leu Ala Gln 1 5 10 15 Leu Glu Trp Lys Asn Gln Ala Leu Glu Lys Lys Leu Ala Gln 20 25 30 <210> 80 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Jun <400> 80 Ile Ala Arg Leu Glu Glu Lys Val Lys Thr Leu Lys Ala Gln Asn Ser 1 5 10 15 Glu Leu Ala Ser Thr Ala Asn Met Leu Arg Glu Gln Val Ala Gln Leu 20 25 30 <210> 81 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> <400> 81 Leu Lys Glu Lys Glu Lys Leu Leu Asn Ala Ile Glu Thr Gln Leu Ala 1 5 10 15 Ser Lys Glu Asp Glu Leu Gln Asp Thr Glu Ala Gln Leu Thr Asp Thr 20 25 30 <210> 82 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> L2 <400> 82 Ser Gly Gly Thr Ser Gly Ser Thr Ser Gly Thr Gly Ser Thr 1 5 10

Claims (28)

A retroviral vector into which a gene encoding a histone or leucine zipper is introduced. The retroviral vector according to claim 1, wherein the gene encoding the histone or leucine zipper is introduced into a gene encoding an integrase. The retroviral vector according to claim 2, wherein the gene encoding the integrase comprises the nucleotide sequence of SEQ ID NO: 83. The retroviral vector according to claim 1, wherein the histone comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70 to 76. The retroviral vector according to claim 1, wherein the gene encoding the histone comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 52 to 58. The retroviral vector according to claim 1, wherein the histone comprises a first linker at the C-terminus, the N-terminus or both ends. The retroviral vector according to claim 1, wherein the gene encoding the histone comprises a gene encoding the first linker at 3', 5' or both. The method of claim 1, wherein the leucine zipper comprises a NZ sequence comprising the amino acid sequence of SEQ ID NO: 78 and a CZ sequence comprising the amino acid sequence of SEQ ID NO: 79; or a Jun sequence comprising the amino acid sequence of SEQ ID NO: 80 and a Fos sequence comprising the amino acid sequence of SEQ ID NO: 81. The method of claim 8, wherein the gene encoding the NZ sequence is a nucleotide sequence including the nucleotide sequence of SEQ ID NO: 60, and the gene encoding the CZ sequence is a nucleotide sequence including the nucleotide sequence of SEQ ID NO: 61, Retrovirus vector. The method of claim 8, wherein the gene encoding the Jun sequence is a nucleotide sequence including the nucleotide sequence of SEQ ID NO: 62, and the gene encoding the Fos sequence is a nucleotide sequence including the nucleotide sequence of SEQ ID NO: 63, Retrovirus vector. The retroviral vector according to claim 1, wherein the leucine zipper comprises a second linker at the C-terminus, the N-terminus or both ends. The method of claim 8, wherein the NZ sequence and the CZ sequence; or wherein the Jun sequence and the Fos sequence are linked via a second linker. The retroviral vector according to claim 2, wherein the gene encoding the histone or the gene encoding the leucine zipper is inserted after position 108 or 816 of the gene encoding integrase. The retroviral vector according to claim 2, wherein the histone or leucine zipper is inserted before residues 37 or 273 of integrase. A method for constructing a retroviral vector into which a gene encoding a histone or leucine zipper is introduced, comprising the steps of:
A gene preparation step of preparing a gene encoding a histone or leucine zipper; and
An introduction step in which a gene is introduced into a retrovirus. The method according to claim 15, wherein the introducing step is to introduce a gene encoding a histone or leucine zipper into a gene encoding an integrase. The method of claim 16, wherein the gene encoding the integrase comprises the nucleotide sequence of SEQ ID NO: 83. The method of claim 15 , wherein the histone comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 65 to 71. The method of claim 15, wherein the gene encoding the histone comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 52 to 58. The method of claim 15 , wherein the histone comprises a first linker at the C-terminus, the N-terminus or both ends. The method of claim 15 , wherein the gene encoding the histone comprises a gene encoding the first linker at 3', 5' or both. 16. The method of claim 15, wherein the leucine zipper comprises a NZ sequence comprising the amino acid sequence of SEQ ID NO: 73 and a CZ sequence comprising the amino acid sequence of SEQ ID NO: 74; or a Jun sequence comprising the amino acid sequence of SEQ ID NO: 75 and a Fos sequence comprising the amino acid sequence of SEQ ID NO: 76. The method of claim 22, wherein the gene encoding the NZ sequence is a nucleotide sequence including the nucleotide sequence of SEQ ID NO: 60, and the gene encoding the CZ sequence is a nucleotide sequence including the nucleotide sequence of SEQ ID NO: 61, Way. The method of claim 22, wherein the gene encoding the Jun sequence is a nucleotide sequence including the nucleotide sequence of SEQ ID NO: 62, and the gene encoding the Fos sequence is a nucleotide sequence including the nucleotide sequence of SEQ ID NO: 63, Way. 16. The method of claim 15, wherein the leucine zipper comprises a second linker at the C-terminus, the N-terminus or both ends. 23. The method of claim 22, wherein the NZ sequence and the CZ sequence; or the Jun sequence and the Fos sequence are linked via a second linker. The method according to claim 15, wherein the gene encoding the histone or leucine zipper is introduced after position 108 or 816 of the gene encoding integrase. The method of claim 15, wherein the histone or leucine zipper is introduced before residues 37 or 273 of integrase.

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